Novel DNA-binding proteins and uses thereof

ABSTRACT

Disclosed herein are polypeptides, polynucleotides encoding, cells and organisms comprising novel DNA-binding domains, including TALE DNA-binding domains. Also disclosed are methods of using these novel DNA-binding domains for modulation of gene expression and/or genomic editing of endogenous cellular sequences.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. ProvisionalApplication Nos. 61/395,836, filed May 17, 2010; 61/401,429, filed Aug.12, 2010; 61/455,121, filed Oct. 13, 2010; 61/459,891, filed Dec. 20,2010; 61/462,482, filed Feb. 2, 2011; 61/465,869, filed Mar. 24, 2011,the disclosures of which are hereby incorporated by reference in theirentireties.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

Not applicable.

FIELD OF THE INVENTION

The present invention provides methods for genetic modification andregulation of expression status of endogenous genes and other genomicloci using engineered DNA binding proteins.

BACKGROUND OF THE INVENTION

Many, perhaps most, physiological and pathophysiological processes canbe controlled by the selective up or down regulation of gene expression.Examples of pathologies that might be controlled by selective regulationinclude the inappropriate expression of proinflammatory cytokines inrheumatoid arthritis, under-expression of the hepatic LDL receptor inhypercholesterolemia, over-expression of proangiogenic factors andunder-expression of antiangiogenic factors in solid tumor growth, toname a few. In addition, pathogenic organisms such as viruses, bacteria,fungi, and protozoa could be controlled by altering gene expression oftheir host cell. Thus, there is a clear unmet need for therapeuticapproaches that are simply able to up-regulate beneficial genes anddown-regulate disease causing genes.

In addition, simple methods allowing the selective over- andunder-expression of selected genes would be of great utility to thescientific community. Methods that permit the regulation of genes incell model systems, transgenic animals and transgenic plants would findwidespread use in academic laboratories, pharmaceutical companies,genomics companies and in the biotechnology industry.

Gene expression is normally controlled through alterations in thefunction of sequence specific DNA binding proteins called transcriptionfactors. They act to influence the efficiency of formation or functionof a transcription initiation complex at the promoter. Transcriptionfactors can act in a positive fashion (activation) or in a negativefashion (repression).

Transcription factor function can be constitutive (always “on”) orconditional. Conditional function can be imparted on a transcriptionfactor by a variety of means, but the majority of these regulatorymechanisms depend of the sequestering of the factor in the cytoplasm andthe inducible release and subsequent nuclear translocation, DNA bindingand activation (or repression). Examples of transcription factors thatfunction this way include progesterone receptors, sterol responseelement binding proteins (SREBPs) and NF-kappa B. There are examples oftranscription factors that respond to phosphorylation or small moleculeligands by altering their ability to bind their cognate DNA recognitionsequence (Hou et al., Science 256:1701 (1994); Gossen & Bujard, Proc.Nat'l Acad Sci 89:5547 (1992); Oligino et al., Gene Ther. 5:491-496(1998); Wang et al., Gene Ther. 4:432-441 (1997); Neering et al., Blood88:1147-1155 (1996); and Rendahl et al., Nat. Biotechnol. 16:757-761(1998)).

Recombinant transcription factors comprising the DNA binding domainsfrom zinc finger proteins (“ZFPs”) have the ability to regulate geneexpression of endogenous genes (see, e.g., U.S. Pat. Nos. 6,534,261;6,599,692; 6,503,717; 6,689,558; 7,067,317; 7,262,054). Clinical trialsusing these engineered transcription factors containing zinc fingerproteins have shown that these novel transcription factors are capableof treating various conditions. (see, e.g., Yu et al. (2006) FASEB J.20:479-481).

Another major area of interest in genome biology, especially in light ofthe determination of the complete nucleotide sequences of a number ofgenomes, is the targeted alteration of genome sequences. Such targetedcleavage events can be used, for example, to induce targetedmutagenesis, induce targeted deletions of cellular DNA sequences, andfacilitate targeted recombination at a predetermined chromosomal locus.See, for example, United States Patent Publications 20030232410;20050208489; 20050026157; 20050064474; 20060188987; 2008015996, andInternational Publication WO 2007/014275, the disclosures of which areincorporated by reference in their entireties for all purposes. See,also, Santiago et al. (2008) Proc Natl Acad Sci USA 105:5809-5814; Perezet al. (2008) Nat Biotechnol 26:808-816 (2008).

Artificial nucleases, which link the cleavage domain of a nuclease to adesigned DNA-binding protein (e.g., zinc-finger protein (ZFP) linked toa nuclease cleavage domain such as from FokI), have been used fortargeted cleavage in eukaryotic cells. For example, zinc fingernuclease-mediated genome editing has been shown to modify the sequenceof the human genome at a specific location by (1) creation of adouble-strand break (DSB) in the genome of a living cell specifically atthe target site for the desired modification, and by (2) allowing thenatural mechanisms of DNA repair to “heal” this break.

To increase specificity, the cleavage event is induced using one or morepairs of custom-designed zinc finger nucleases that dimerize uponbinding DNA to form a catalytically active nuclease complex. Inaddition, specificity has been further increased by using one or morepairs of zinc finger nucleases that include engineered cleavagehalf-domains that cleave double-stranded DNA only upon formation of aheterodimer. See, e.g., U.S. Patent Publication No. 20080131962,incorporated by reference herein in its entirety.

The double-stranded breaks (DSBs) created by artificial nucleases havebeen used, for example, to induce targeted mutagenesis, induce targeteddeletions of cellular DNA sequences, and facilitate targetedrecombination at a predetermined chromosomal locus. See, for example,United States Patent Publications 20030232410; 20050208489; 20050026157;20050064474; 20060188987; 20060063231; 20070218528; 20070134796;20080015164 and International Publication Nos. WO 07/014,275 and WO2007/139982, the disclosures of which are incorporated by reference intheir entireties for all purposes. Thus, the ability to generate a DSBat a target genomic location allows for genomic editing of any genome.

There are two major and distinct pathways to repair DSBs—homologousrecombination and non-homologous end-joining (NHEJ). Homologousrecombination requires the presence of a homologous sequence as atemplate (known as a “donor”) to guide the cellular repair process andthe results of the repair are error-free and predictable. In the absenceof a template (or “donor”) sequence for homologous recombination, thecell typically attempts to repair the DSB via the error-prone process ofNHEJ.

The plant pathogenic bacteria of the genus Xanthomonas are known tocause many diseases in important crop plants. Pathogenicity ofXanthomonas depends on a conserved type III secretion (T3S) system whichinjects more than 25 different effector proteins into the plant cell.Among these injected proteins are transcription activator-like effectors“TALE” or “TAL-effectors”) which mimic plant transcriptional activatorsand manipulate the plant transcriptome (see Kay et al (2007) Science318:648-651). These proteins contain a DNA binding domain and atranscriptional activation domain. One of the most well characterizedTALEs is AvrBs3 from Xanthomonas campestris pv. Vesicatoria (see Bonaset al (1989) Mol Gen Genet 218: 127-136 and WO2010079430). TALEs containa centralized repeat domain that mediates DNA recognition, with eachrepeat unit containing approximately 33-35 amino acids specifying onetarget base. TALEs also contain nuclear localization sequences andseveral acidic transcriptional activation domains (for a review seeSchornack S, et al (2006) J Plant Physiol 163(3): 256-272). In addition,in the phytopathogenic bacteria Ralstonia solanacearum two genes,designated brg11 and hpx17 have been found that are homologous to theAvrBs3 family of Xanthomonas in the R. solanacearum biovar 1 strainGMI1000 and in the biovar 4 strain RS1000 (See Heuer et al (2007) Appland Envir Micro 73(13): 4379-4384). These genes are 98.9% identical innucleotide sequence to each other but differ by a deletion of 1,575 bpin the repeat domain of hpx17. However, both gene products have lessthan 40% sequence identity with AvrBs3 family proteins of Xanthomonas.

DNA-binding specificity of these TALEs depends on the sequences found inthe tandem TALE repeat units. The repeated sequence comprisesapproximately 33-35 amino acids and the repeats are typically 91-100%homologous with each other (Bonas et al, ibid). There appears to be aone-to-one correspondence between the identity of the hypervariablediresidues at positions 12 and 13 with the identity of the contiguousnucleotides in the TALE's target sequence (see Moscou and Bogdanove,(2009) Science 326:1501 and Boch et al (2009) Science 326:1509-1512).These two adjacent amino acids are referred to as the Repeat VariableDiresidue (RVD). Experimentally, the natural code for DNA recognition ofthese TALEs has been determined such that an HD sequence at positions 12and 13 leads to a binding to cytosine (C), NG binds to T, NI to A, NNbinds to G or A, and NG binds to T. These specificity-determining TALErepeat units have been assembled into proteins with new combinations ofthe natural TALE repeat units and altered numbers of repeats, to makevariant TALE proteins. When in their native architecture, these variantsare able to interact with new sequences and activate the expression of areporter gene in plant cells (Boch et al., ibid.). However, theseproteins maintain the native (full-length) TALE protein architecture andonly the number and identity of the TALE repeat units within theconstruct were varied. Entire or nearly entire TALE proteins have alsobeen fused to a nuclease domain from the FokI protein to create aTALE-nuclease fusion protein (“TALEN”), and these TALENs have been shownto cleave an episomal reporter gene in yeast cells. (Christian et al.(2010) Genetics 186(2): 757-61; Li et al. (2011a) Nucleic Acids Res.39(1):359-372). Such constructs could also modify endogenous genes inyeast cells to quantifiable levels and could modify endogenous genes inmammalian and plant cells to detectable, but unquantifiable levels whenappropriate sequence amplification schemes are employed. See, Li et al.(2011b) Nucleic Acids Res. epub doi:10.1093/nar/gkr188; Cermak et al.(2011) Nucleic Acids Res. epub doi:10.1093/nar/gkr218. The fact that atwo step enrichment scheme was required to detect activity in plant andanimal cells indicates that fusions between nearly entire TALE proteinsand the nuclease domain from the FokI protein do not efficiently modifyendogenous genes in plant and animal cells. In other words, the peptideused in these studies to link the TALE repeat array to the FokI cleavagedomain does not allow efficient cleavage by the Fold domain ofendogenous genes in higher eukaryotes. These studies therefore highlightthe need to develop compositions that can be used connect a TALE arraywith a nuclease domain that would allow for highly active cleavage inendogenous eukaryotic settings.

There remains a need for engineered DNA binding domains to increase thescope, specificity and usefulness of these binding proteins for avariety of applications including engineered transcription factors forregulation of endogenous genes in a variety of cell types and engineerednucleases that can be similarly used in numerous models, diagnostic andtherapeutic systems, and all manner of genome engineering and editingapplications.

SUMMARY OF THE INVENTION

The present invention thus provides for methods of targeted manipulationof expression state or sequence of endogenous loci. In some embodimentsof the invention, the methods of the invention use DNA-binding proteinscomprising one or more TALE-repeat units fused to functional proteindomains (collectively “TALE-fusions”), to form engineered transcriptionfactors, engineered nucleases (“TALENs”), recombinases, transposases,integrases, methylases, enzymatic domains and reporters. In someaspects, the polypeptide includes the at least one TALE repeat unitlinked to additional TALE protein sequences, for efficient and specificfunction at endogenous target DNA. These additional sequences, which arelinked to the N- and optionally the C-termini of the TALE repeat domain,are also referred to as the “N-cap” and “C-cap” sequences. Thus, theinvention provides polypeptides comprising one or more (e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20 or more) TALE repeat and/orhalf-repeat units.

Thus, in one aspect, provided herein is a DNA-binding polypeptidecomprising at least one TALE repeat unit (e.g., 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more repeat unit(s)).The polypeptide typically includes an N-cap sequence (polypeptide) ofany length that supports DNA-binding function of the TALE repeat(s) orfunctional activity of the TALE fusion protein. Optionally, thepolypeptide may also include a C-cap sequence (polypeptide), for examplea C-cap sequence of less than approximately 250 amino acids (C+230C-cap; from residue C-20 to residue C+230). In addition, in certainembodiments, at least one of the TALE repeat units of the TALEpolypeptides as described herein include repeat variable di-residue(RVD) regions that are atypical. The TALE repeat unit may be a wild-typedomain isolated from Xanthomonas, Ralstonia or another related bacteriaand/or may be engineered in some manner (e.g., may be non-naturallyoccurring). In certain embodiments, at least one TALE repeat unit isengineered (e.g., non-naturally occurring, atypical, codon optimized,combinations thereof, etc.). In certain embodiments, one or more aminoacids in the TALE repeat domain (e.g., an RVD within one of the TALErepeats) are altered such that the domain binds to a selected targetsequence (typically different from the target sequence bound by anaturally occurring TALE DNA binding domain). In other embodiments, atleast one TALE repeat unit is modified at some or all of the amino acidsat positions 4, 11, 12, 13 or 32 within the TALE repeat unit. In someembodiments; at least one TALE repeat unit is modified at 1 or more ofthe amino acids at positions 2, 3, 4, 11, 12, 13, 21, 23, 24, 25, 26,27, 28, 30, 31, 32, 33, 34, or 35 within one TALE repeat unit. In otherembodiments, the nucleic acid encoding the TALE repeat is modified suchthat the DNA sequence is altered but the amino acid sequence is not. Insome embodiments, the DNA modification is for the purposes of codonoptimization. In further embodiments, at least one TALE repeat unit isaltered by combinations of the above described modifications. In someembodiments, TALE proteins comprising several modified TALE repeat unitsare provided. Combinations of naturally occurring and non-naturallyoccurring TALE repeat units are also provided. In a preferredembodiment, the TALE protein (wild-type or engineered) further comprisesN-cap and optionally the C-cap sequences for efficient and specificfunction at endogenous target DNA. In some embodiments, the N-capcomprises residues N+1 to N+136 (see FIG. 1B for a description of theresidue numbering scheme), or any fragment thereof. In otherembodiments, the C-cap comprises residues C-20 to C+28, C-20 to C+39,C-20 to C+55, or C-20 to C+63 or any fragments of the full length TALEC-terminus thereof. In certain embodiments, the polypeptide comprisingthe TALE repeat domain, as well as an N-cap and optional C-capsequences, further comprises a regulatory or functional domain, forexample, a transcriptional activator, transcriptional repressor,nuclease, recombinase, transposase, integrase, methylase or the like.

Polynucleotides encoding these proteins are also provided as arepharmaceutical compositions. In addition, the invention includes hostcells, cell lines and transgenic organisms (e.g., plants, fungi,animals) comprising these proteins/polynucleotides and/or modified bythese proteins (e.g., genomic modification that is passed onto theprogeny). Exemplary cells and cell lines include animal cells (e.g.,mammalian, including human, cells such as stem cells), plant cells,bacterial cells, protozoan cells, fish cells, or fungal cells. Inanother embodiment, the cell is a mammalian cell. Methods of making andusing these proteins and/or polynucleotides are also provided.

In one aspect, provided herein are fusion proteins comprising one ormore engineered TALE repeat units, an N-cap, and an optional C-capsequence, operatively linked to one or more heterologous polypeptidedomains, for example functional (regulatory) domains. Librariescomprising modules of TALE repeats are provided as are optionalstructured or flexible linkers for connecting the engineered TALErepeats to the functional protein domain of interest. The functionalprotein domain (e.g., transcriptional activator, repressor, or nuclease)may be positioned at the C- or N-termini of the fusion protein. Methodsof making fusion proteins as described herein are also provided.

The present invention also provides a method for identifying suitabletarget sequences (sites) for engineered TALE fusion proteins. In someembodiments, a target site identified has an increased number of guaninenucleotides (“G”) as compared to a natural TALE target sequence. Inother embodiments, the target does not require flanking thymidinenucleotides (“T”), as typical in naturally occurring TALE proteins. Insome embodiments, the RVDs selected for use in the engineered TALEprotein contains one or more NK (asparagine-lysine) RVDs for therecognition of G nucleotides in the target sequence. Additionallyprovided in this invention are novel (non-naturally occurring) RVDs,differing from those found in nature, which are capable of recognizingnucleotide bases. Non-limiting examples of atypical or non-naturallyoccurring RVDs (amino acid sequences at positions 12 and 13 of the TALErepeat unit) include RVDs as shown in Table 30A, for example, VG and IAto recognize T, RG to recognize A and T, and AA to recognize A, C, and Tare provided. Also provided are RVDs capable of interacting equally withall nucleotide bases (e.g. A, C, T, and G). Additional RVDs useful inthe compositions and methods described herein are shown in Table 27.

Also provided by the invention are methods to constrain, or notconstrain, by the user's choice, the distance or gap spacing between thetwo target sites on a nucleic acid that is subject to modification by aTALE-nuclease (“TALEN”) heterodimer. In some embodiments, the gapspacing is constrained to 12-13 base pairs, while in other embodiments,the engineered TALEN is designed to cleave DNA targets comprising a gapspacing of between 12 to 21 base pairs. In some embodiments, the TALENheterodimer is designed to cleave a sequence comprising a gap of between1 and 34 nucleotides between each monomer binding site. In still moreembodiments, the TALEN is constrained to cleave a target with a 12 or 13base pair gap by utilizing a TALEN architecture comprising the +28C-terminal truncation (C+28 C-cap). In other embodiments, the designedTALEN is made to cleave a target nucleic acid comprising a 12 to 21 basepair gap spacing using a TALEN architecture comprising the +63C-terminal truncation, which increases the likelihood of being able toidentify a suitable TALEN target site due to the flexibility in gapspacing requirements. In some embodiments, the TALEN has an engineeredR1/2 repeat such that the R1/2 repeat is capable of targeting nucleotidebases other than T.

In another aspect, the present invention provides a vector for anengineered TALE DNA binding domain fusion wherein the vector comprisesthe TALE N-cap and C-cap sequences flanking the TALE repeat sequences aswell as locations to allow for the cloning of multiple TALE repeatunits, linker sequences, promoters, selectable markers, polyadenylationsignal sites, functional protein domains and the like. Also provided bythe invention herein is a method for the construction of a modulararchive library including at least one TALE-repeat unit (e.g.,engineered) for ready assembly of specific TALE DNA binding domaindomains and fusion proteins comprising these domains (e.g., TALENs).

In yet another aspect, the present invention provides a method ofmodulating the expression of an endogenous cellular gene in a cell, themethod comprising the step of: contacting a first target site in theendogenous cellular gene with a first engineered TALE fused to afunctional domain (e.g., transcriptional modulator domain), therebymodulating expression of the endogenous cellular gene. In anotheraspect, the present invention provides a method of modulating expressionof an endogenous cellular gene in a cell, the method comprising the stepof: contacting a target site in the endogenous cellular gene with afusion TALE protein wherein the TALE comprises an engineered TALE repeatdomain such that the TALE has specificity for a desired sequence. Insome embodiments, the modulatory effect is to activate the expression ofthe endogenous gene. In some embodiments, the expression of theendogenous gene is inhibited. In yet another embodiment, activation orrepression of the endogenous gene is modulated by the binding of a TALEfusion protein such that an endogenous activator or repressor cannotbind to the regulator regions of the gene of interest.

In one embodiment, the step of contacting further comprises contacting asecond target site in an endogenous cellular gene with a secondengineered TALE fusion protein, thereby modulating expression of thesecond endogenous cellular gene. In another embodiment, the first andsecond target sites are adjacent. In certain embodiments, the first andsecond target sites are in different genes, for example to modulateexpression of two or more genes using TALE-transcription factors. Inother embodiments, the first and second target sites are in the samegene, for example when a pair of TALEN fusion proteins is used to cleavein the same gene. The first and second target sites are separated by anyof base pairs (“gap size”), for example, 1 to 20 (or any numbertherebetween) or even more base pairs. In another embodiments, the stepof contacting further comprises contacting more than two target sites.In certain embodiments, two sets of target sites are contacted by twopairs of TALENs, and are used to create a specific deletion or insertionat the two sets of targets. In another embodiment, the first TALEprotein is a fusion protein comprising a regulatory or functionaldomain. In another, embodiment, the first TALE protein is a fusionprotein comprising at least two regulatory or functional domains. Inanother embodiment, the first and second TALE proteins are fusionproteins, each comprising a regulatory domain. In another embodiment,the first and second TALE proteins are fusion proteins, each comprisingat least two regulatory domains. The one or more functional domains maybe fused to either (or both) ends of the TALE protein. Any of the TALEfusions proteins can be provided as polynucleotides encoding theseproteins.

In yet another aspect, the invention provides compositions for C-capslinking a nuclease domain to a TALE repeat domain as described herein,wherein the resulting fusion protein exhibits highly active nucleasefunction. In some embodiments the C-cap comprises peptide sequence fromnative TALE C-terminal flanking sequence. In other embodiments, theC-cap comprises peptide sequence from a TALE repeat domain. In yetanother embodiment, the C-cap comprises sequences not derived from TALEproteins. C-caps may also exhibit a chimeric structure, for examplecomprising peptide sequences from native TALE C-terminal flankingsequence and/or TALE repeat domains and/or non-TALE polypeptides.

In any of the compositions or methods described herein, the regulatoryor functional domain may be selected from the group consisting of atranscriptional repressor, a transcriptional activator, a nucleasedomain, a DNA methyl transferase, a protein acetyltransferase, a proteindeacetylase, a protein methyltransferase, a protein deaminase, a proteinkinase, and a protein phosphatase. In some aspects, the functionaldomain is an epigenetic regulator. In plants, such a TALE fusion can beremoved by out-crossing using standard techniques. In such anembodiment, the fusion protein would comprise an epigenetic regulatorsuch as, by non-limiting example, a histone methyltransferase, DNAmethyltransferase, or histone deacetylase. See for example, co-ownedU.S. Pat. No. 7,785,792.

Thus, in some aspects, the TALE fusion protein comprises a TALE-repeatdomain fused to a nuclease domain (a “TALEN”). As noted above, in someembodiments the TALE repeat domain is further fused to an N-cap sequenceand, optionally, a C-cap sequence. In other embodiments, the nucleasedomain is connected to either the amino terminus of the N-cap or carboxyterminus of the C-cap via linker peptide sequences that provideefficient catalytic function of the nuclease domain. The nuclease domainmay be naturally occurring or may be engineered or non-naturallyoccurring. In some embodiments, the nuclease domain is derived from aType IIS nuclease (e.g. FokI). In other embodiments, the TALE DNAbinding domain is operably linked to a Bfi I nuclease domain. In someembodiments, the Fold domain is a single chain nuclease domain,comprising two cleavage half domains, and in others it is a FokIcleavage half domain. In some aspects of the invention, a single TALENprotein is used by itself to induce a double strand break in a targetDNA, while in others, the TALEN is used as part of a pair of nucleases.In some embodiments, the pair comprises two TALENs comprising FokI halfdomains, wherein the pairing of the FokI half domains is required toachieve DNA cleavage, while in other cases the TALEN protein is used incombination with a zinc-finger nuclease wherein pairing of the two Foldcleavage domains is required to achieve DNA cleavage. In someembodiments, the TALE DNA binding domain is fused to a zinc finger tomake a zin finger/TALE hybrid DNA binding domain. In some instances, thehybrid DNA binding domain is able to skip interacting with internalstretches of DNA bases within the DNA target binding site. In someembodiments, the FokI domains are able to form homodimers, and in otherinstances, heterodimerization of two non-identical FokI cleavage domainsfrom each member of the TALEN pair is required for targeted cleavageactivity. In these heterodimeric TALEN pairs, two Fold domains of thesame type are not able to productively homodimerize. In otherembodiments, a TALEN pair is used wherein one FokI cleavage domain isinactive such that pairing may occur, but the target DNA is nicked toproduce a cut on one strand of the DNA molecule rather than cleavingboth strands.

In any of the compositions or methods described herein, the TALE fusionprotein may be encoded by a TALE fusion protein nucleic acid. In certainembodiments, the sequence encoding the TALE fusion protein is operablylinked to a promoter. Thus, in certain embodiments, the methods ofmodulating endogenous gene expression or genomic modification furthercomprises the step of first administering the nucleic acid encoding theTALE protein to the cell. The TALE-fusion protein may be expressed froman expression vector such as a retroviral expression vector, anadenoviral expression vector, a DNA plasmid expression vector, or an AAVexpression vector. In some embodiments, the expression vector is alentiviral vector, and in some of these embodiments, the lentiviralvector is integrase-defective.

Also provided in the invention are TALENs (e.g., TALEN pairs) specificto any desired target locus (e.g., endogenous gene) in any cell type.Non-limiting examples include TALENs specific for NTF3, VEGF, CCR5,IL2Rγ, BAX, BAK, FUT8, GR, DHFR, CXCR4, GS, Rosa26, AAVS1 (PPP1R12C),MHC genes, PITX3, ben-1, Pou5F1 (OCT4), C1, RPD1, etc.

The TALE-repeat domains as described herein may bind to a target sitethat is upstream of, or adjacent to, a transcription initiation site ofthe endogenous cellular gene. Alternatively, the target site may beadjacent to an RNA polymerase pause site downstream of a transcriptioninitiation site of the endogenous cellular gene. In still furtherembodiments, the TALE fusion protein (e.g., a TALEN) binds to a sitewithin the coding sequence of a gene or in a non-coding sequence withinor adjacent to the gene; such as for example, a leader sequence, trailersequence or intron, or within a non-transcribed region, either upstreamor downstream of the coding region.

In another aspect, described herein is a method for cleaving one or moregenes of interest in a cell, the method comprising: (a) introducing,into the cell, one or more one or more TALEN protein(s) (orpolynucleotides encoding the TALENs) that bind to a target site in theone or more genes under conditions such that the TALEN protein(s) is(are) expressed and the one or more genes are cleaved. In embodiments inwhich two or more TALEN proteins are introduced, one, some or all can beintroduced as polynucleotides or as polypeptides. In some aspects, saidgene cleavage results in the functional disruption of the targeted gene.Cleavage of the targeted DNA may be followed by NHEJ wherein smallinsertions or deletions (indels) are inserted at the site of cleavage:These indels then cause functional disruption through introduction ofnon-specific mutations at the cleavage location.

In yet another aspect, described herein is a method for introducing anexogenous sequence into the genome of a cell, the method comprising thesteps of: (a) introducing, into the cell, one or more TALEN protein(s)(or polynucleotides encoding the TALEN protein(s)) that bind to a targetsite in a target gene under conditions such that the TALEN protein(s) is(are) expressed and the one or more target sites within the genes arecleaved; and (b) contacting the cell with an exogenous polynucleotide;such that cleavage of the DNA target site(s) stimulates integration ofthe exogenous polynucleotide into the genome by homologousrecombination. In certain embodiments, the exogenous polynucleotide isintegrated physically into the genome. In other embodiments, theexogenous polynucleotide is integrated into the genome by copying of theexogenous sequence into the host cell genome via specialized nucleicacid replication processes associated with homology-directed repair(HDR) of the double strand break. In yet other embodiments, integrationinto the genome occurs through non-homology dependent targetedintegration (e.g. “end-capture”). In some embodiments, the exogenouspolynucleotide comprises a recombinase recognition site (e.g. loxP orFLP) for recognition by a cognate recombinase (e.g. Cre or FRT,respectively). In certain embodiments, the exogenous sequence isintegrated into the genome of a small animal (e.g. rabbit or rodent suchas mouse, rat, etc.). In one embodiment, the TALE-fusion proteincomprises a transposase, recombinase or integrase, wherein theTALE-repeat domain has been engineered to recognize a specificallydesired target sequence. In some embodiments, TALE polypeptides areused. In some aspects, the TALE-fusion protein comprises a tranposase orintegrase and is used for the development of a CHO-cell specifictransposase/integrase system.

In some embodiments, the TALE-fusion protein comprises amethyltransferase wherein the TALE-repeat domain has been engineered torecognize a specifically desired target sequence. In some embodiments,the TALE-repeat domain is fused to a subunit of a protein complex thatfunctions to effect epigenetic modification of the genome or ofchromatin.

In yet further embodiments, that TALE-fusion further comprises areporter or selection marker wherein the TALE-repeat domain has beenengineered to recognize a specifically desired target sequence. In someaspects, the reporter is a fluorescent marker, while in other aspects,the reporter is an enzyme.

In another aspect, described herein are compositions comprising one ormore of the TALE-fusion proteins. In certain embodiments, thecomposition comprises one or more TALE-fusion proteins in combinationwith a pharmaceutically acceptable excipient. In some embodiments, thecomposition comprises a polynucleotide encoding the TALE fusion protein.Some embodiments comprise a composition comprising a DNA moleculeencoding a TALEN. In other embodiments, the composition comprises a RNAmolecule encoding a TALEN. Some compositions further comprise a nucleicacid donor molecule.

In another aspect, described herein is a polynucleotide encoding one ormore TALE-fusion proteins described herein. The polynucleotide may be,for example, mRNA.

In another aspect, described herein is a TALE-fusion protein expressionvector comprising a polynucleotide, encoding one or more TALE-fusionproteins described herein, operably linked to a promoter (e.g.,constitutive, inducible, tissue-specific or the like).

In another aspect, described herein is a host cell comprising one ormore TALE-fusion proteins and/or one or more polynucleotides (e.g.,expression vectors encoding TALE-fusion proteins as described herein. Incertain embodiments, the host cell further comprises one or more zincfinger proteins and/or ZFP encoding vectors. The host cell may be stablytransformed or transiently transfected or a combination thereof with oneor more of these protein expression vectors. In other embodiments, theone or more protein expression vectors express one or fusion proteins inthe host cell. In another embodiment, the host cell may further comprisean exogenous polynucleotide donor sequence. Any prokaryotic oreukaryotic host cells can be employed, including, but not limited to,bacterial, plant, fish, yeast, algae, insect, worm or mammalian cells.In some embodiments, the host cell is a plant cell. In other aspects,the host cell is part of a plant tissue such as the vegetative parts ofthe plant, storage organs, fruit, flower and/or seed tissues. In furtherembodiments, the host cell is an algae cell. In other embodiments, thehost cell is a fibroblast. In any of the embodiments, described herein,the host cell may comprise a stem cell, for example an embryonic stemcell. The stem cell may be a mammalian stem cell, for example, ahematopoietic stem cell, a mesenchymal stem cell, an embryonic stemcell, a neuronal stem cell, a muscle stem cell, a liver stem cell, askin stem cell, an induced pluripotent stem cell and/or combinationsthereof. In certain embodiments, the stem cell is a human inducedpluripotent stem cells (hiPSC) or a human embryonic stem cell (hESC). Inany of the embodiments, described herein, the host cell can comprise anembryo cell, for example one or more mouse, rat, rabbit or other mammalcell embryos. In some aspects, stem cells or embryo cells are used inthe development of transgenic animals, including for example animalswith TALE-mediated genomic modifications that are integrated into thegermline such that the mutations are heritable. In further aspects,these transgenic animals are used for research purposes, i.e. mice,rats, rabbits; while in other aspects, the transgenic animals arelivestock animals, i.e. cows, chickens, pigs, sheep etc. In stillfurther aspects, the transgenic animals are those used for therapeuticpurposes, i.e. goats, cows, chickens, pigs; and in other aspects, thetransgenic animals are companion animals, i.e. cats, dogs, horses, birdsor fish.

Another aspect provided by the invention is a method for identifying asuitable nucleic acid target for TALE binding. In some embodiments, atarget is chosen based upon its similarity to target sites used bytypical, naturally occurring TALE proteins. In other embodiments, atarget is selected that is not utilized by typical, naturally occurringTALE proteins because the engineered TALE proteins have been altered insuch a way as to make them able to interact with an atypical, targetsequence. In some embodiments, this alteration involves the selection ofatypical (non-naturally occurring or rare) RVD sequences. In furtherembodiments, the atypical RVD used is a ‘NK’ RVD for the recognition ofa G residue in the desired target sequence. In other embodiments,targets are selected that contain non-natural ratios of nucleic acidbases because the engineered TALE proteins have been altered in such away as to make them able to interact with a non-natural ratio of nucleicacid bases. In some embodiments, the ratio of bases in the desiredtarget sequence comprises an unusual number of G residues. In otherembodiments, the ratio of bases in the desired target sequence comprisesan unusual number of atypical di-nucleotides, tri-nucleotides ortetra-nucleotides. Further provided are design rules for identifying themost optimal targets for TALE-DNA binding interactions. These rulesprovide guidance on selection of a target site sequence comprisingoptimal di- and tri-nucleotide pairs. In addition, these rules alsoprovide guidance on less optimal di- and tri-nucleotide pairs so thatthe artisan may avoid these sequences if desired. Also provided are RVDsable to interact with all nucleotides to provide the user a greaterflexibility in choosing target sequences.

In one aspect, the invention provides compositions and methods for invivo genomic manipulation. In certain embodiments, mRNAs encoding TALENsmay be injected into gonads, ovum or embryos for introducing specificDSBs as desired. In some embodiments, donor nucleotides are co-deliveredwith the TALEN mRNAs to cause specific targeted integration in theorganism.

In yet a further aspect, provided herein are kits comprising theTALE-domain proteins (and fusion proteins comprising these TALE-repeatproteins) of the invention. These kits may be used to facilitate genomicmanipulation by the user and so can provide a TALEN, for example, thatwill cleave a desired target or a safe harbor locus within a genome. TheTALEN may be provided either as nucleic acid (e.g. DNA or RNA) or may beprovided as protein. In some instances, the protein may be formulated toincrease stability, or may be provided in a dried form. In someinstances, the kits are used for diagnostic purposes. In some instances,the TALE-fusion included in the kit is a transcriptional regulator. Insome instances, the TALE-fusion comprises a reporter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1, panels A and B, depict a TALE protein. FIG. 1A shows a schematicof the domain structure of a TALE protein (not drawn to scale). ‘N’ and‘C’ indicate the amino and carboxy termini, respectively. The TALErepeat domain, N-cap and C-cap are labeled and the residue numberingscheme for the N-cap and C-cap in this protein are indicated. “R0”represents the 34 amino acids preceding the first tandem TALE repeatthat may share some structural homology with the TALE repeat units andthat may specify thymine in a DNA target sequence. “R_(1/2)” denotes theC-terminal TALE “half-repeat,” which is a 20 residue peptide sequence(with residues numbered from C−20 to C−1) with homology to the first 20residues of a typical TALE repeat. NLS is the nuclear localizationsequence. AD is the acidic activation domain. FIG. 1B (SEQ ID NO:135)shows the primary sequence of a cloned natural TALE protein (hereinafterreferred to as “TALE13”) that was isolated with a cloning schemedesigned to delete the N-terminal 1-152 amino acid residues. The N-capand C-cap are indicated by a thick black line below the sequence;positions N+1 and N+136 in the N-cap and positions C+1 and C+278 in theC-cap are indicated. The half repeat is the first 20 residues of theC-cap and ends immediately prior to the position indicated as “C+1”.Underlined residues in the TALE repeats and half repeat indicate aminoacids (RVDs) that specify the DNA nucleotide contacted by the repeatduring target binding.

FIG. 2, panels A and B, show the reporter construct for use with thepredicted target of TALE13 (TR13). FIG. 2A (SEQ ID NO:136) shows aschematic of the reporter vector indicating the cloning sites used forinserting 1-4 TR13 targets into the vector. The region in italics is thepromoter region for the luciferase gene. FIG. 2B (SEQ ID NO:137) showsthe linker sequence used containing two TR13 targets.

FIG. 3, panels A and B, show a schematic of the reporter constructcontaining 0-4 TR13 targets (FIG. 3A) and synergistic reporter geneactivation by TALE13-VP16 fusion protein (TR13-VP16, TALE13 linked withan activation domain from VP16) on the luciferase reporter constructscontaining 1 to 4 multiple TR13 targets, indicated as R13x1 to R13x4,respectively (FIG. 3B). pGL3 is the control reporter vector lacking anyTR13 target elements.

FIG. 4, panels A and B, show reporter gene activation by TALE VP16fusion proteins. FIG. 4A is a schematic of the TALE proteins, with orwithout the addition of VP16 domain, as well as the reporter constructsused in the study. R13x2 indicates the construct where two of the TALE13(TR13) targets are inserted while R15x2 indicates the construct wheretwo of the TALE15 (TR15) targets are inserted. FIG. 4B shows thereporter gene activation by TALE protein with the VP16 fusion but not bythe TALE protein itself. Thus, the natural transcriptional activationdomain present in the TALE protein was not functional in mammalian cellsin this assay. Moreover, the transcriptional activity observed wasspecific as the reporter gene activation occurs only when the correcttargets are matched with their corresponding TALE VP16 fusions. Thecloned TALE13 and TALE15 are indicated as TR13 and TR15 respectively.TR13-VP16 and TR15-VP16 are similar to TR13 and TR15 with the additionalVP16 activation domain fused to their C-terminus.

FIG. 5, panels A and B, depict positional effects of target sequenceplacement relative to the promoter. FIG. 5A shows a schematic of thereporter constructs where the target sequences are placed eitherproximal (R13x4) or distal (R13x4D) to the SV40 promoter. FIG. 5B showsthe reporter gene activation by the indicated TALEs. “nR13V-d145C”refers to an expression construct containing the SV40 nuclearlocalization sequence, the TR13 sequence with 145 amino acid residuesdeleted from the C-terminus (yielding a C+133 C-cap) and the VP16activation domain, whereas “R13-VP16” refers to an expression constructcontaining TALE13 sequence and the VP16 activation domain. As shown, (i)the C-terminal 145 amino acids of the full length TALEs are not requiredfor the reporter gene activation, and (ii) the reporter gene activationis greatest when the target sequences are placed proximal to thepromoter sequence.

FIG. 6, panels A and B, are graphs depicting the reporter gene(luciferase) activation using a TALE fusion. FIG. 6A depicts theactivation of a reporter gene using a fusion protein comprising theengineered TALE 18 protein (R23570 here; referred to as NT-L in laterfigures). The reporter construct contains 2 copies of the engineeredTALE18 targets upstream from the luciferase gene. Activation of thisreporter is observed only with R23570V, which contains the 17.5engineered repeat sequences (17 full TALE repeats and one half repeat),the N- and C-terminal sequences (N-cap and C-cap) flanking the tandemTALE repeats of TR13, and the VP16 activation domain. Deletion of boththe N- and C-terminal flanking sequences (N-cap and C-cap) abolishes theactivity (compare nR23570S-dNC to mock). nR23570S-dNC contains the SV40NLS (n), the 17.5 engineered TALE repeat sequences, fused to a singlep65 activation domain (S), but is lacking the N- and C-terminalsequences (N-cap and C-cap) from TALE (dNC). The nR23570SS-dNC is thesame as nR23570S-dNC except that it has two p65 domains. The R0-VP16construct is the same as R23570 but lacks the tandem TALE repeats.‘Mock’ shows the results for an experiment lacking an expressionconstruct. FIG. 6B depicts the activation of an endogenous gene in itschromosomal environment by a fusion protein comprising the engineered(non-naturally occurring) TALE18 domain. The engineered TALE18(R23570V), which is designed to target to the NTF3 gene, can lead to asubstantial increase in the endogenous NTF3 mRNA level. Under the sameconditions, the expression of NTF3 mRNA is not affected by eitherR0-VP16 or GFP. R23570V and R0-VP16 are described as above.

FIG. 7, panels A to D, depict additional exemplary NTF3-specific TALEtranscription factor fusions. FIG. 7A depicts a diagram of the exemplaryproteins and their target in the NTF3 promoter (SEQ ID NO:138). The twoTALE transcription factor variants were linked to the VP16 activationdomain and expressed in HEK293 cells. The sequence at the bottom showsthe promoter-proximal region of human NTF3. Underlined bases indicatethe target site for the NT-L TALE repeat domain. The hooked arrow showsthe start site of NTF3 transcription. FIG. 7B shows relative NTF3 mRNAlevels in HEK293 cells expressing either the top or lower proteinsketched in FIG. 7A. “eGFP” indicates cells transfected with a controlplasmid that expresses enhanced GFP. Measurements were performed inquadruplicate and error bars indicate standard deviations. FIG. 7Cdepicts levels of NTF3 protein secreted from HEK293 cells expressingeither the top or lower proteins sketched in 7A. Measurements wereperformed in duplicate using an ELISA assay, and error bars indicatestandard deviations. “Neg.” indicates cells transfected with an emptyvector control. FIG. 7D shows the RVDs (top row of letters), expectedbinding site (second row of letters) and SELEX-derived base frequencymatrix for NT-L (graph at bottom). Except for the first and fifthpositions in the matrix, the most frequently selected base matches thetarget locus sequence.

FIG. 8, panels A and B, are graphs depicting the DNA binding ability, asassayed by ELISA, of a series of N- and C-terminal truncations ofvarious engineered TALE DNA binding domains. FIG. 8A depicts the datafor an NT3-specific TALE DNA binding domain comprising 9.5 TALE repeats,while FIG. 8B depicts the data for a VEGF-specific TALE DNA bindingdomain comprising 9.5 TALE repeats. For both sets of data, when theN-terminal truncations were made, the C-terminus was maintained at theC+95 position while for the C-terminal truncations, the N-terminus wasmaintained at the N+137 position (these constructs have a methionineresidue appended to the N+136 N-cap residue). As can be seen, bothproteins showed an apparent decrease in relative DNA binding affinityunder the conditions of this assay when the protein was truncated on theN-terminus further than the N+134 position. Additionally, both proteinsshowed an apparent decrease in relative DNA binding affinity under theconditions of this assay when the C-terminus was truncated past aminoacid C+54.

FIG. 9, panels A and B, depict the DNA binding activity, as assayed byELISA, of a series of N- and C-terminal truncations as described above.In FIG. 9A, the data for the NTF3-specific TALE DNA binding domain isshown, but in this case, when the N-terminal truncations were beingtested, the C-terminus was maintained at the C+54 position. For theC-terminal truncations, the N-terminal amino acid was the N+134position. In FIG. 9B, the data for the VEGF-specific TALE DNA bindingdomains is shown. As shown, the N- and C-terminal ends were maintainedas described above for FIG. 9A.

FIG. 10 shows dissection of TALE functional domains involved foractivity. The activities for reporter gene activation by indicatedconstructs as illustrated in Table 16 were investigated. The resultsindicate that (i) the N-terminal 152 amino acids and C-terminal 183amino acids are not required for robust function in this assay; and (ii)the sequence flanking the tandem TALE repeats, including R0 region andthe leucine rich domain, restore the functional activity in cells inthis assay. Deletion of either N-terminal sequence preceding the firstTALE repeat or C-terminal sequences following the last repeat abolishesfunctional activity in this assay. R13V-d145C has a C+133 C-cap,R13V-d182C has a C+95 C-cap, R13V-dC has a C+22 C-cap, nR13V-dN has aN+8 N-cap, nR13V-d223N has an N+52 N-cap and nR13V-d240 has an N+34N-cap.

FIG. 11, panels A and B, depict nuclease activity of TALE13 linked totwo copies of the Fold domain in K562 cells. FIG. 11A depicts aschematic of a single stranded annealing based reporter assay (SSA) fordetecting the nuclease activity in mammalian cells. The reporterconstruct (SSA-R13) in this assay contained the TALE13 target,sandwiched by the N-terminal (GF) and C-terminal part (FP) of the GFPcoding sequence. The plasmid SSA-R13 by itself cannot drive the GFPexpression, but the cleavage of the R13 target promotes homologousrecombination between the N-terminal (GF) and C-terminal (FP) part ofthe GFP to form a functional GFP. Thus, the nuclease activity of TALENprotein was assessed by analyzing the percentage of the GFP positivecells. FIG. 11B demonstrates nuclease activity by a TALEN protein. TheGFP positive cells generated from SSA-R13 reporter construct increasedsignificantly using a TALEN (R13d182C-scFokI; C+95 C-cap), compared to acontrol experiment lacking the nuclease plasmid (mock). R13d182C-scFokIis the same as R13V-d182C described above except that two copies of FokIdomain, linked by 12 copies of GGGGS sequences between the FokI domains,is used to replace the VP16 activation domain.

FIG. 12 depicts an ethidium bromide gel showing nuclease activity of theTALE-13 effector domain-FokI cleavage half-domain fusions in vitro. Thecolumns show data for four TALE domain nuclease cleavage proteins: thenuclease fusion with a N+137, C+28 configuration using either the L2 orL8 linker (see Example 7); the nuclease fusion with the N+137, C+39configuration, using the L2 linker; and the N+137, C+63 fusion with theL2 linker. The gap spacings between the two target sites are shownbeneath the wells where the number indicates the number of by betweenthe targets. “S” indicates a single target site for only one half of thepair. “PmlI” indicates cleavage with a standard restriction enzyme andblank indicates the results when the experiment was carried out withoutthe nuclease encoding plasmid.

FIG. 13 is a graph depicting the DNA cleavage obtained by the indicatedTALE13-FokI cleavage half domain fusions. “Dimer Gap” indicates thenumber of by between the two target sites, and “Percent DNA Cleavage”indicates how much DNA was cleaved in the reaction. The results indicatethat virtually 100 percent DNA cleavage is achievable in these reactionconditions with the three of the four nucleases tested.

FIG. 14 depicts an ethidium bromide-stained gel showing nucleaseactivity of the TALE domain-FokI half cleavage domain fusions. In thisexperiment, the N-terminus was varied while the C-terminus wasmaintained with the C+63 configuration. The Pml1 and Blank controls arethe same as for FIG. 12. The N-terminal truncations tested in thisexperiment were N+137, N+134, N+130 and N+119. The different DNA targetsites are indicated as in FIG. 12 except that the label is above thecognate lane rather than below it. Activity of the nucleases isdiminished when the N-terminus is shorter than approximately +134 to+137. The amount of DNA loaded in each lane for the 5 bp gap and 8 bpgap targets was uneven so it is difficult to determine if the lowerbands in these lanes represent DNA cleavage products or background bandsdue to inefficient PCR at the inverted repeats.

FIG. 15, panels A and B, depict TALEN activity in K562 cells. FIG. 15A(SEQ ID NO:342) depicts the target sequence used in the reporter plasmidfor the NTF3 targeting TALE pairs which also includes binding sites fora pair of CCR5-specific ZFNs (8267/8196). FIG. 15B is a graph depictingthe results of the SSA nuclease assay where (−)NT3 R18 C28L8 (light graybars; C+28 C-cap, L8 linker) depicts data observed when only one memberof the NTF3-specific pair was present while (+)NT3 R18 C28L8 (dark graybars) depicts the results when both members of the pair were present.“8267EL8196KK” indicates the results using the CCR5-specific ZFN pair.

FIG. 16 depicts the results of a Cel-I Surveyor™ mismatch assay(Transgenomics, “Cel-I assay”) on cells treated with various pairs ofNTF3-targeting TALENs. The samples, numbered 1-30 are as described inthe text. (+) denotes addition of the Cel-I enzyme, (−) denotes theassay without any added enzyme. A band of approximately 226 bp isapparent in most of the samples, indicating a mismatch induced bycleavage of the endogenous NTF3 target by the nuclease, followed bynon-homologous end joining which introduces areas of mismatch with thewild type sequence. “gfp” indicates the control where cells weretransfected with a GFP encoding plasmid only. The percent NHEJ activityquantitated on the gel is indicated in each sample containing the Cel-Ienzyme. The gel demonstrates that the pairs induced targeted locusdisruption at up to 8.66% of total alleles in some samples at thisendogenous locus in a mammalian cell.

FIG. 17, panels A through C, depict the activity of NTF3-specific TALENsin K562 cells. FIG. 17A shows the SELEX specificity data for theengineered TALEN protein designated NT-R which is the engineered partnermade for the NT-L TALEN fusion. The expected bases and correspondingRVDs are shown above the plot. The +63 C-terminal flanking region wasused for this SELEX experiment. FIG. 17B shows a gel of the results of aCel-I assay using four NTF3-specific TALEN pairs in K562 cells where theculture conditions were either at 30° C. or 37° C. As can be seen fromthe data presented, the most active pair demonstrated gene modificationlevels of 3% at 37° C. and 9% under cold-shock conditions (30° C.)(Doyon et al. (2010) Nat Methods 8(1):74-9. Epub 2010 Dec. 5 and U.S.application Ser. No. 12/800,599). 84 amplicons from the PCR pool fromthe cold-shock study were then sequenced, and seven mutated alleles wereidentified, which are shown in FIG. 17C (SEQ ID NO:343-350). As can beseen, small indels are observed.

FIG. 18, panels A and B, depict the sequencing results observedfollowing endogenous cleavage of the NTF3 locus in K562 cells usingTALENs. FIG. 18A depicts the chromosomal sequence (SEQ ID NO:139-140)and the boxes delineate the binding sites for the two TALENs. FIG. 18Bdepicts a compilation of sequencing results of the NTF3 locus from cellstreated with the different NTF3 TALEN pairs described in Example 8aligned with the wild-type (“wt”) sequence (SEQ ID NO:141-175).

FIG. 19 depicts the results of a targeted integration event at anendogenous gene via a DSB induced by the NTF3-specific TALENs.Oligonucleotides for capture in the DSB were synthesized to containoverhangs corresponding to all possible sequences within the spacebetween the TALEN binding sites. PCR was done using a set of primersthat primed off of the inserted oligonucleotide and a region outside theputative cut site. Eight (8) different pairs of NTF3-specific TALENswere tested wherein the pairs are labeled A-H. The legend shows aportion of the gel demonstrating how the lanes are read.

FIG. 20, panels A to D, show capture of an oligonucleotide duplex at anendogenous chromosomal locus mediated by NHEJ following a DSB induced atthat locus by a TALEN pair. FIG. 20A shows part of the NTF3 target locus(top duplex, SEQ ID NO:351) and one of the oligonucleotide duplexes usedfor this study (bottom duplex, SEQ ID NO:352). Binding sites for NT-L+28and NT-R+63 are underlined in the top sequence. The cleavage overhangthat will most efficiently capture the duplex (5′ CTGG) is alsohighlighted. FIG. 20B shows part of the NTF3 target locus (top duplex,SEQ ID NO:353) and the second oligonucleotide duplex used for this study(bottom sequences, SEQ ID NO:354). Binding sites for NT-L+28 and NT-R+63are underlined in the top sequence. The cleavage overhang that will mostefficiently capture this second duplex (5′ TGGT) is also shown. FIG. 20C(SEQ ID NO:355-357) shows results following expression of NT-L+28 andNT-R+63 in K562 cells in the presence of the oligonucleotide duplexshown in FIG. 20A. Junctions between successfully integrated duplex andgenomic DNA were then amplified using one primer that anneals within theduplex and one primer that anneals to the native NTF3 locus. Theresulting amplicons were cloned and sequenced. The “expected” sequenceat top indicates the sequence that would result from a perfect ligationof oligonucleotide duplex to the cleaved locus. The box highlights thelocation of the duplex overhang in the junction sequences. The bottomtwo lines provide junction sequences obtained from this study. As shown,eleven junction sequences resulted from perfect ligation of duplex tothe cleavage overhang, while one junction sequence exhibited a shortdeletion (12 bp) consistent with resection prior to repair by NHEJ. FIG.20D (SEQ ID NO:358-362) shows results from experiments as shown in FIG.20C except that the oligonucleotide duplex shown in FIG. 20B was used,which has a 4 bp overhang that is shifted by one base relative to theduplex shown in FIG. 20A. The lowest four lines provide junctionsequences obtained from this study. As shown, four distinct sequenceswere identified, which each exhibit short deletions consistent withresection prior to NHEJ-mediated repair.

FIG. 21 depicts several of the potential secondary DNA structurespredicted to form in the natural TALE repeat domain during PCRamplification that can disrupt efficient amplification of the template.Analysis of the DNA sequence of the TALE-repeat protein was done usingMfold (M. Zuker Nucleic Acids Res. 31(13):3406-15, (2003)). 800 basepairs of the nucleic acid sequence were analyzed starting at the 5′ endof the nucleic acid encoding the first full TALE repeat sequence. Thesequence analyzed contained approximately 7.5 repeats. Analysis revealedseveral very stable secondary, structures.

FIG. 22 depicts pictoral results of in silico analysis of 1963 TALErepeats from Xanthomonas bacteria displaying the conserved amino acidsat each position in the 34 amino acid repeat unit. Letter size isinversely related to observed diversity at any given position: largerletters indicate less tolerance of diversity while smaller lettersindicate the alternate amino acids that can be observed at a givenlocation. Different shades of color represent different chemical classesof the amino acids. In this sample of 1963 TALE repeats, the mostfrequency RVDs were: 28.8% HD; 20.6% NI, 15.1% NN; 13.2% NG; 8.5% NS;5.5% HG; and 5.5% NG* (where the asterisk indicates the RVD was observedin a 33-residue TALE repeat instead of the more typical 34-residuerepeat). 15 other RVD sequences were observed in this sample, but theseall had frequencies below 1%.

FIG. 23 depicts a schematic of the method used to tandemly link PCRamplicons of selected TALE repeat modules and ligate them into a vectorbackbone to create the desired TALE fusion protein. Specific primers arelisted in Example 11. Also depicted is the vector backbone into whichthe assembled TALE fusion is cloned. The fusion partner domain is a FokInuclease catalytic domain to allow production of one member of a TALENpair.

FIG. 24, panels A and B, depict the use of TALENs to drivehomology-based transfer of a short segment of heterology encoding a RFLPinto the endogenous CCR5 locus. FIG. 24A shows a schematic for theassay, and depicts the location of the PCR primers used and the Bgl Isite. FIG. 24B depicts a gel showing insertion of a 46 bp donor sequenceinto a DSB introduced by a CCR5-specific TALEN pair. The donor sequencecontains a unique BglI restriction site, so upon PCR amplification ofthe target site and then digestion of the PCR product with BglI,sequences that have been cleaved by the TALEN pair and have hadinsertion of the 46 bp donor sequence will have two BglI cleavageproducts, as indicated in the Figure.

FIG. 25, panels A and B, are graphs depicting the cleavage efficacy ofTALENs as compared to target gap spacings. FIG. 25A depicts the activityof a panel of CCR5-specific TALEN pair with a +28/+28 pairing (C+28C-cap on both TALENs) while FIG. 25B depicts the activity of a panelCCR5-specific TALEN pair comprising a +63/+63 pairing (C+63 C-cap onboth TALENs). As can be seen, the activity of the +28/+28 pair is moretightly constrained to a 12 or 13 bp gap spacing between the two targetsequences while the +63/+63 pair exhibits activity across a gap spacingrange of 12-23 bp.

FIG. 26 is a graph depicting the endogenous activity of a CCR5-specificTALEN pair with different length C-cap sequences, or stated another way,different sequences linking the array of full TALE repeats to thenuclease domain. C terminal truncations were made across the C-terminalsequence to yield C-caps from C-2 to C+278. These constructs were testedfor TALEN activity in K562 cells against an endogenous target with an 18bp gap spacing where the cells were incubated at either 37° C. (lightsquares) or cold shock conditions (30° C., dark diamonds). The activitywas highly dependent on the identity of the sequence used to connect thearray of full TALE repeats with the FokI cleavage domain. Note that ourC-cap notation does not include C+0 so the C−1 C-cap value was plottedat X=0 and C−2 was plotted as X=−1. C+5, C+28, etc. were plotted as X=5,X=28, etc. Peak activity was observed for a C+63 C-cap sequence.

FIG. 27 depicts the specificity of an exemplary TALEN chosen for RVDanalysis. The TALEN was designed to bind to the 11 base target sequence5′-TTGACAATCCT-3′ (SEQ ID NO:178). Shown are the DNA binding resultsdetermine by ELISA analysis when this target is altered at position 6,such that the identity of the target at positions 5-7 is either CAA(designed target), CGA, TCG or TTG.

FIG. 28 is a graphical display of the ELISA affinities measured for allthe RVDs tested. The data are shown in a 20×20 grid where the firstamino acid of the RVD (position 12) is indicated on the vertical left ofthe grid and the second amino acid of the RVD (position 13) is indicatedhorizontally above the grid. The size of the letters A, C, G, and T ineach grid is scaled based on the square root of the normalized ELISAsignal for the CAA site, CCA site, and CGA site and CTA siterespectively. Many RVDs have improved DNA binding properties withrespect to the naturally occurring HD, NI, NG, NS, NN, IG, HG, and NKRVDs. The four RVDs that are the most frequently found in nature (HD,NG, NI, and NN) are boxed for reference. For these four RVDs, thepreferred base by ELISA matched expected preferred base.

FIG. 29 are gels depicting the results of measurements of activity ofTALENs in which the C-terminal half repeat has been altered at the RVDto allow interaction with nucleotide bases other than T. Shown TALENactivities as determined by Cel-I assay as described above. Arrow headsindicate bands that are a result of Cel-I cleavage at indels. Laneassignments are as listed in Example 16, Table 32. These resultsdemonstrate that TALEN C-terminal half repeats can be engineered to bindto each nucleotide base as desired.

FIG. 30 are gels depicting the measurement of TALEN activity usingTALENs that have TALE repeat units comprising either fully atypical RVDs(Fully Substituted), repeat domains where all the repeat units of onetype or specificity have been substituted (e.g. all repeat units withRVDs that specify ‘T’ etc.) with atypical RVDs (Type Substitutions), orTALENs where only one repeat unit with the array has been substitutedwith an atypical RVD-comprising repeat unit (Singly Substituted).Activity assays were carried out either at 37 degrees or under coldshock conditions (30 degrees), and quantitation of any measurable NHEJactivity is indicated on the lanes.

FIG. 31 is a series of gels depicting the presence of NHEJ events in ratpups born following TALEN treatment of rat embryos. Genomic DNA wasisolated from the pups and PCR was performed on the region surroundingthe nuclease target site. The product was then examined for NHEJ inducedmismatches using the T7 endonuclease. The arrow indicates the band thatis produced from the presence of a mismatch. 7 of 66 pups examined (11%)were positive for an NHEJ event.

DETAILED DESCRIPTION OF THE INVENTION

Introduction

The present application demonstrates that TALE-repeat domains can beengineered to recognize a desired endogenous DNA sequence and thatfusing functional domains to such engineered TALE-repeat domains can beused to modify the functional state, or the actual genomic DNA sequenceof an endogenous cellular locus, including a gene, that is present inits native chromatin environment. The present invention thus providesTALE-fusion DNA binding proteins that have been engineered tospecifically recognize, with high efficacy, endogenous cellular lociincluding genes. As a result, the TALE-fusions of the invention can beused to regulate endogenous gene expression, both through activation andrepression of endogenous gene transcription. The TALE-fusions can alsobe linked to other regulatory or functional domains, for examplenucleases, transposases or methylases, to modify endogenous chromosomalsequences.

The methods and compositions described herein allow for novel human andmammalian therapeutic applications, e.g., treatment of genetic diseases,cancer, fungal, protozoal, bacterial, and viral infection, ischemia,vascular disease, arthritis, immunological disorders, etc., as well asproviding for functional genomics assays, and generating engineered celllines for research and drug screening, and means for developing plantswith altered phenotypes, including but not limited to, increased diseaseresistance, and altering fruit ripening characteristics, sugar and oilcomposition, yield, and color.

As described herein, two or more TALE-fusions can be administered to anycell, recognizing either the same target endogenous cellular gene, ordifferent target endogenous cellular genes.

In another embodiment, the TALE-fusion protein is linked to at least oneor more regulatory domains, described below. Non-limiting examples ofregulatory or functional domains include transcription factor repressoror activator domains such as KRAB and VP16, co-repressor andco-activator domains, DNA methyl transferases, histoneacetyltransferases, histone deacetylases, and DNA cleavage domains suchas the cleavage domain from the endonuclease FokI.

Described herein are also compositions and methods including fusionproteins comprising one or more TALE-repeat units, an N-cap and,optionally, a C-cap fused to nuclease domains useful for genomic editing(e.g., cleaving of genes; alteration of genes, for example by cleavagefollowed by insertion (physical insertion or insertion viahomology-directed repair) of an exogenous sequence and/or cleavagefollowed by NHEJ; partial or complete inactivation of one or more genes;generation of alleles with altered functional states of endogenousgenes, insertion of regulatory elements; etc.) and alterations of thegenome which are carried into the germline. Also disclosed are methodsof making and using these compositions (reagents), for example to edit(alter) one or more genes in a target cell. Thus, the methods andcompositions described herein provide highly efficient methods fortargeted gene alteration (e.g., knock-in) and/or knockout (partial orcomplete) of one or more genes and/or for randomized mutation of thesequence of any target allele, and, therefore, allow for the generationof animal models of human diseases.

Also disclosed herein are compositions (C-caps) for linking a nucleasedomain to a TALE repeat array that provide highly active nucleasefunction. In some embodiments the C-cap comprises peptide sequence froma native TALE C-terminal flanking sequence. In other embodiments, theC-cap comprises peptide sequence from a TALE repeat domain. In yetanother embodiment the C-cap comprises non-TALE sequences. C-caps mayalso exhibit a chimeric structure, containing peptide sequences fromnative TALE C-terminal flanking sequence and/or TALE repeat domainsand/or neither of these sources.

TALENs can also be engineered to allow the insertion of a donor ofinterest into a safe harbor locus such as AAVS1 (see co-owned US PatentPublication 20080299580) or CCR5 (see co-owned United States PatentPublication 20080159996). The donor can comprise a gene of interest orcan encode an RNA of interest such as an shRNA, RNAi or miRNA.

The expression of engineered TALE-fusion proteins (e.g., transcriptionalactivators, transcriptional repressors and nucleases) can be alsocontrolled by systems typified by the tet-regulated systems and theRU-486 system (see, e.g., Gossen & Bujard, Proc Natl Acad Sci 89:5547(1992); Oligino et al., Gene Ther. 5:491-496 (1998); Wang et al., GeneTher. 4:432-441 (1997); Neering et al, Blood 88:1147-1155 (1996); andRendahl et al., Nat. Biotechnol. 16:757-761 (1998)). These impart smallmolecule control on the expression of the TALE-fusion activators andrepressors and thus impart small molecule control on the target gene(s)of interest. This beneficial feature could be used in cell culturemodels, in gene therapy, and in transgenic animals and plants.

General

Practice of the methods, as well as preparation and use of thecompositions disclosed herein employ, unless otherwise indicated,conventional techniques in molecular biology, biochemistry, chromatinstructure and analysis, computational chemistry, cell culture,recombinant DNA and related fields as are within the skill of the art.These techniques are fully explained in the literature. See, forexample, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Secondedition, Cold Spring Harbor Laboratory Press, 1989 and Third edition,2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley& Sons, New York, 1987 and periodic updates; the series METHODS INENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE ANDFUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS INENZYMOLOGY, Vol. 304, “Chromatin” (P. M. Wassarman and A. P. Wolffe,eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULARBIOLOGY, Vol. 119, “Chromatin Protocols” (P. B. Becker, ed.) HumanaPress, Totowa, 1999.

DEFINITIONS

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” areused interchangeably and refer to a deoxyribonucleotide orribonucleotide polymer, in linear or circular conformation, and ineither single- or double-stranded form. For the purposes of the presentdisclosure, these terms are not to be construed as limiting with respectto the length of a polymer. The terms can encompass known analogues ofnatural nucleotides, as well as nucleotides that are modified in thebase, sugar and/or phosphate moieties (e.g., phosphorothioatebackbones). In general, an analogue of a particular nucleotide has thesame base-pairing specificity; i.e., an analogue of A will base-pairwith T.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues. The termalso applies to amino acid polymers in which one or more amino acids arechemical analogues or modified derivatives of a correspondingnaturally-occurring amino acids.

“Binding” refers to a sequence-specific, non-covalent interactionbetween macromolecules (e.g., between a protein and a nucleic acid). Notall components of a binding interaction need be sequence-specific (e.g.,contacts with phosphate residues in a DNA backbone), as long as theinteraction as a whole is sequence-specific. Such interactions aregenerally characterized by a dissociation constant (K_(d)) of 10⁻⁶ M orlower. “Affinity” refers to the strength of binding: increased bindingaffinity being correlated with a lower K_(d).

A “binding protein” is a protein that is able to bind non-covalently toanother molecule. A binding protein can bind to, for example, a DNAmolecule (a DNA-binding protein), an RNA molecule (an RNA-bindingprotein) and/or a protein molecule (a protein-binding protein). In thecase of a protein-binding protein, it can bind to itself (to formhomodimers, homotrimers, etc.) and/or it can bind to one or moremolecules of a different protein or proteins. A binding protein can havemore than one type of binding activity. For example, zinc-fingerproteins have DNA-binding, RNA-binding and protein-binding activity.

A “TALE-repeat domain” (also “repeat array”) is a sequence that isinvolved in the binding of the TALE to its cognate target DNA sequenceand that comprises one or more TALE “repeat units.” A single “repeatunit” (also referred to as a “repeat”) is typically 33-35 amino acids inlength and exhibits at least some sequence homology with other TALErepeat sequences within a naturally occurring TALE protein. A TALErepeat unit as described herein is generally of the form(X)^(1 to 11)-(X^(RVD)) ₂-(X)₂₀₋₂₂ (SEQ ID NO:399) where X^(RVD)(positions 12 and 13) exhibit hypervariability in naturally occurringTALE proteins. Altering the identity of the amino acids at positions 12and 13 can alter the preference for the identity of the DNA nucleotide(or pair of complementary nucleotides in double-stranded DNA) with whichthe repeat unit interacts. An “atypical” RVD is an RVD sequence(positions 12 and 13) that occurs infrequently or never in nature, forexample, in less than 5% of naturally occurring TALE proteins,preferably in less than 2% of naturally occurring TALE proteins and evenmore preferably less than 1% of naturally occurring TALE proteins. Anatypical RVD can be non-naturally occurring.

The terms “N-cap” polypeptide and “N-terminal sequence” are used torefer to an amino acid sequence (polypeptide) that flanks the N-terminalportion of the TALE repeat domain. The N-cap sequence can be of anylength (including no amino acids), so long as the TALE-repeat domain(s)function to bind DNA. Thus, an N-cap sequence may be involved insupplying proper structural stabilization for the TALE repeat domainand/or nonspecific contacts with DNA. An N-cap sequence may be naturallyoccurring or non-naturally occurring, for example it may be derived fromthe N-terminal region of any full length TALE protein. The N-capsequence is preferably a fragment (truncation) of a polypeptide found infull-length TALE proteins, for example any truncation of a N-terminalregion flanking the TALE repeat domain in a naturally occurring TALEprotein that is sufficient to support DNA-binding function of theTALE-repeat domain or provide support for TALE fusion protein activity.When each TALE-repeat unit comprises a typical RVD and/or when the C-capcomprises a full-length naturally occurring C-terminal region of a TALEprotein, the N-cap sequence does not comprise a full-length N-terminalregion of a naturally occurring TALE protein. Thus, as noted above, thissequence is not necessarily involved in DNA recognition, but may enhanceefficient and specific function at endogenous target DNA or efficientactivity of the TALE fusion protein. The portion of the N-cap sequenceclosest to the N-terminal portion of the TALE repeat domain may bearsome homology to a TALE repeat unit and is referred to as the “R0repeat.” Typically, the preferred nucleotide to the position immediately5′ of the target site is thymidine (T). It may be that the R0 repeatportion of the N-cap prefers to interact with a T (or the A base-pairedto the T in double-stranded DNA) adjacent to the target sequencespecified by the TALE repeats. Shown below is one example of an R0sequence:

LDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLN (SEQ ID NO:1)

The term “C-cap” or “C-terminal region” refers to optionally presentamino acid sequences (polypeptides) that may be flanking the C-terminalportion of the TALE repeat domain. The C-cap can also comprise any partof a terminal C-terminal TALE repeat, including 0 residues, truncationsof a TALE repeat or a full TALE repeat. The first 20 residues of theC-terminal region are typically homologous to the first 20 residues of aTALE repeat unit and may contain an RVD sequence capable of specifyingthe preference of nucleotides 3′ of the DNA sequence specified by theTALE repeat domain. When present, this portion of the C-terminal regionhomologous to the first 20 residues of a TALE repeat is also referred toas the “half repeat.” The numbering scheme of residues in the C-terminalregion reflects this typical partial homology where the number schemestarts at C−20, increments to C−19, C−18, C−17, C−16, C−15, C−14, C−13,C−12, C−11, C−10, C−9, C−8, C−7, C−6, C−5, C−4, C−3, C−2, C−1,increments to C+1, and then increments to C+2, C+3, etc. towards theC-terminus of the polypeptide. A C+28 C-cap refers to the sequence fromresidue C−20 to residue C+28 (inclusive) and thus has a length of 48residues. The C-cap sequences may be naturally occurring (e.g.,fragments of naturally occurring proteins) or non-naturally occurring(e.g., a fragment of a naturally occurring protein comprising one ormore amino acid deletions, substitutions and/or additions), or any othernatural or non-natural sequence with the ability to act as a C cap. TheC-terminal region is not absolutely required for the DNA-bindingfunction of the TALE repeat domain(s), but, in some embodiments, a C-capmay interact with DNA and also may enhance the activity of functionaldomains, for example in a fusion protein comprising a nuclease at theC-terminal to the TALE repeat domain.

A “zinc-finger DNA binding protein” (or binding domain) is a protein, ora domain within a larger protein, that binds DNA in a sequence-specificmanner through one or more zinc-fingers, which are regions of amino acidsequence within the binding domain whose structure is stabilized throughcoordination of a zinc ion. The term zinc-finger DNA binding protein isoften abbreviated as zinc-finger protein or ZFP.

A “selected” zinc-finger protein or protein comprising a TALE-repeatdomain is a protein whose production results primarily from an empiricalprocess such as phage display, interaction trap or hybrid selection. Seee.g., U.S. Pat. No. 5,789,538; U.S. Pat. No. 5,925,523; U.S. Pat. No.6,007,988; U.S. Pat. No. 6,013,453; U.S. Pat. No. 6,200,759; WO95/19431; WO 96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO01/60970 WO 01/88197 and WO 02/099084.

The term “sequence” refers to a nucleotide sequence of any length, whichcan be DNA or RNA; can be linear, circular or branched and can be eithersingle-stranded or double stranded. The term “donor sequence” refers toa nucleotide sequence that is inserted into a genome. A donor sequencecan be of any length, for example between 2 and 10,000 nucleotides inlength (or any integer value therebetween or thereabove), preferablybetween about 100 and 1,000 nucleotides in length (or any integertherebetween), more preferably between about 200 and 500 nucleotides inlength.

A “homologous, non-identical sequence” refers to a first sequence whichshares a degree of sequence identity with a second sequence, but whosesequence is not identical to that of the second sequence. For example, apolynucleotide comprising the wild-type sequence of a mutant gene ishomologous and non-identical to the sequence of the mutant gene. Incertain embodiments, the degree of homology between the two sequences issufficient to allow homologous recombination therebetween, utilizingnormal cellular mechanisms. Two homologous non-identical sequences canbe any length and their degree of non-homology can be as small as asingle nucleotide (e.g., for correction of a genomic point mutation bytargeted homologous recombination) or as large as 10 or more kilobases(e.g., for insertion of a gene at a predetermined ectopic site in achromosome). Two polynucleotides comprising the homologous non-identicalsequences need not be the same length. For example, an exogenouspolynucleotide (i.e., donor polynucleotide) of between 20 and 10,000nucleotides or nucleotide pairs can be used.

Techniques for determining nucleic acid and amino acid sequence identityare known in the art. Typically, such techniques include determining thenucleotide sequence of the mRNA for a gene and/or determining the aminoacid sequence encoded thereby, and comparing these sequences to a secondnucleotide or amino acid sequence. Genomic sequences can also bedetermined and compared in this fashion. In general, identity refers toan exact nucleotide-to-nucleotide or amino acid-to-amino acidcorrespondence of two polynucleotides or polypeptide sequences,respectively. Two or more sequences (polynucleotide or amino acid) canbe compared by determining their percent identity. The percent identityof two sequences, whether nucleic acid or amino acid sequences, is thenumber of exact matches between two aligned sequences divided by thelength of the shorter sequences and multiplied by 100.

Alternatively, the degree of sequence similarity between polynucleotidescan be determined by hybridization of polynucleotides under conditionsthat allow formation of stable duplexes between homologous regions,followed by digestion with single-stranded-specific nuclease(s), andsize determination of the digested fragments. Two nucleic acid, or twopolypeptide sequences are substantially homologous to each other whenthe sequences exhibit at least about 70%-75%, preferably 80%-82%, morepreferably 85%-90%, even more preferably 92%, still more preferably 95%,and most preferably 98% sequence identity over a defined length of themolecules, as determined using the methods above. As used herein,substantially homologous also refers to sequences showing completeidentity to a specified DNA or polypeptide sequence. DNA sequences thatare substantially homologous can be identified in a Southernhybridization experiment under, for example, stringent conditions, asdefined for that particular system. Defining appropriate hybridizationconditions is within the skill of the art. See, e.g., Sambrook et al.,supra; Nucleic Acid Hybridization: A Practical Approach, editors B. D.Hames and S. J. Higgins, (1985) Oxford; Washington, D.C.; IRL Press).

“Recombination” refers to a process of exchange of genetic informationbetween two polynucleotides. For the purposes of this disclosure,“homologous recombination (HR)” refers to the specialized form of suchexchange that takes place, for example, during repair of double-strandbreaks in cells via homology-directed repair mechanisms. This processrequires nucleotide sequence homology, uses a “donor” molecule totemplate repair of a “target” molecule (i.e., the one that experiencedthe double-strand break), and is variously known as “non-crossover geneconversion” or “short tract gene conversion,” because it leads to thetransfer of genetic information from the donor to the target. Withoutwishing to be bound by any particular theory, such transfer can involvemismatch correction of heteroduplex DNA that forms between the brokentarget and the donor, and/or “synthesis-dependent strand annealing,” inwhich the donor is used to resynthesize genetic information that willbecome part of the target, and/or related processes. Such specialized HRoften results in an alteration of the sequence of the target moleculesuch that part or all of the sequence of the donor polynucleotide isincorporated into the target polynucleotide.

In the methods of the disclosure, one or more targeted nucleases asdescribed herein create a double-stranded break in the target sequence(e.g., cellular chromatin) at a predetermined site, and a “donor”polynucleotide, having homology to the nucleotide sequence in the regionof the break, can be introduced into the cell. The presence of thedouble-stranded break (DSB) has been shown to facilitate integration ofthe donor sequence. The donor sequence may be physically integrated or,alternatively, the donor polynucleotide is used as a template for repairof the break via homologous recombination, resulting in the introductionof all or part of the nucleotide sequence as in the donor into thecellular chromatin. Thus, a first sequence in cellular chromatin can bealtered and, in certain embodiments, can be converted into a sequencepresent in a donor polynucleotide. Thus, the use of the terms “replace”or “replacement” can be understood to represent replacement of onenucleotide sequence by another, (i.e., replacement of a sequence in theinformational sense), and does not necessarily require physical orchemical replacement of one polynucleotide by another. In someembodiments, two DSBs are introduced by the targeted nucleases describedherein, resulting in the deletion of the DNA in between the DSBs. Insome embodiments, the “donor” polynucleotides are inserted between thesetwo DSBs.

Thus, in certain embodiments, portions of the donor sequence that arehomologous to sequences in the region of interest exhibit between about80 to 99% (or any integer therebetween) sequence identity to the genomicsequence that is replaced. In other embodiments, the homology betweenthe donor and genomic sequence is higher than 99%, for example if only 1nucleotide differs as between donor and genomic sequences of over 100contiguous base pairs. In certain cases, a non-homologous portion of thedonor sequence can contain sequences not present in the region ofinterest, such that new sequences are introduced into the region ofinterest. In these instances, the non-homologous sequence is generallyflanked by sequences of 50-1,000 base pairs (or any integral valuetherebetween) or any number of base pairs greater than 1,000, that arehomologous or identical to sequences in the region of interest. In otherembodiments, the donor sequence is non-homologous to the first sequence,and is inserted into the genome by non-homologous recombinationmechanisms.

In any of the methods described herein, additional TALE-fusion proteinsfused to nuclease domains as well as additional pairs of TALE- (or zincfinger) nucleases can be used for additional double-stranded cleavage ofadditional target sites within the cell.

Any of the methods described herein can be used for partial or completeinactivation of one or more target sequences in a cell by targetedintegration of donor sequence that disrupts expression of the gene(s) ofinterest. Cell lines with partially or completely inactivated genes arealso provided.

Furthermore, the methods of targeted integration as described herein canalso be used to integrate one or more exogenous sequences. The exogenousnucleic acid sequence can comprise, for example, one or more genes orcDNA molecules, or any type of coding or noncoding sequence, as well asone or more control elements (e.g., promoters). In addition, theexogenous nucleic acid sequence may produce one or more RNA molecules(e.g., small hairpin RNAs (shRNAs), inhibitory RNAs (RNAis), microRNAs(miRNAs), etc.).

“Cleavage” refers to the breakage of the covalent backbone of a DNAmolecule. Cleavage can be initiated by a variety of methods including,but not limited to, enzymatic or chemical hydrolysis of a phosphodiesterbond. Both single-stranded cleavage and double-stranded cleavage arepossible, and double-stranded cleavage can occur as a result of twodistinct single-stranded cleavage events. DNA cleavage can result in theproduction of either blunt ends or staggered ends. In certainembodiments, fusion polypeptides are used for targeted double-strandedDNA cleavage.

A “cleavage half-domain” is a polypeptide sequence which, in conjunctionwith a second polypeptide (either identical or different) forms acomplex having cleavage activity (preferably double-strand cleavageactivity). The terms “first and second cleavage half-domains;” “+ and −cleavage half-domains” and “right and left cleavage half-domains” areused interchangeably to refer to pairs of cleavage half-domains thatdimerize.

An “engineered cleavage half-domain” is a cleavage half-domain that hasbeen modified so as to form obligate heterodimers with another cleavagehalf-domain (e.g., another engineered cleavage half-domain). See, also,U.S. Patent Publication Nos. 2005/0064474; 2007/0218528 and2008/0131962, incorporated herein by reference in their entireties.

“Chromatin” is the nucleoprotein structure comprising the cellulargenome. Cellular chromatin comprises nucleic acid, primarily DNA, andprotein, including histones and non-histone chromosomal proteins. Themajority of eukaryotic cellular chromatin exists in the form ofnucleosomes, wherein a nucleosome core comprises approximately 150 basepairs of DNA associated with an octamer comprising two each of histonesH2A, H2B, H3 and H4; and linker DNA (of variable length depending on theorganism) extends between nucleosome cores. A molecule of histone H1 isgenerally associated with the linker DNA. For the purposes of thepresent disclosure, the term “chromatin” is meant to encompass all typesof cellular nucleoprotein, both prokaryotic and eukaryotic. Cellularchromatin includes both chromosomal and episomal chromatin.

A “chromosome,” is a chromatin complex comprising all or a portion ofthe genome of a cell. The genome of a cell is often characterized by itskaryotype, which is the collection of all the chromosomes that comprisethe genome of the cell. The genome of a cell can comprise one or morechromosomes.

An “episome” is a replicating nucleic acid, nucleoprotein complex orother structure comprising a nucleic acid that is not part of thechromosomal karyotype of a cell. Examples of episomes include plasmidsand certain viral genomes.

A “target site” or “target sequence” is a nucleic acid sequence thatdefines a portion of a nucleic acid to which a binding molecule willbind, provided sufficient conditions for binding exist. For example, thesequence 5′-GAATTC-3′ is a target site for the Eco RI restrictionendonuclease.

“Plant” cells include, but are not limited to, cells of monocotyledonous(monocots) or dicotyledonous (dicots) plants. Non-limiting examples ofmonocots include cereal plants such as maize, rice, barley, oats, wheat,sorghum, rye, sugarcane, pineapple, onion, banana, and coconut.Non-limiting examples of dicots include tobacco, tomato, sunflower,cotton, sugarbeet, potato, lettuce, melon, soybean, canola (rapeseed),and alfalfa. Plant cells may be from any part of the plant and/or fromany stage of plant development.

An “exogenous” molecule is a molecule that is not normally present in acell, but can be introduced into a cell by one or more genetic,biochemical or other methods. “Normal presence in the cell” isdetermined with respect to the particular developmental stage andenvironmental conditions of the cell. Thus, for example, a molecule thatis present only during embryonic development of muscle is an exogenousmolecule with respect to an adult muscle cell. Similarly, a moleculeinduced by heat shock is an exogenous molecule with respect to anon-heat-shocked cell. An exogenous molecule can comprise, for example,a functioning version of a malfunctioning endogenous molecule or amalfunctioning version of a normally-functioning endogenous molecule. Anexogenous molecule can also be a molecule normally found in anotherspecies, for example, a human sequence introduced into an animal'sgenome.

An exogenous molecule can be, among other things, a small molecule, suchas is generated by a combinatorial chemistry process, or a macromoleculesuch as a protein, nucleic acid, carbohydrate, lipid, glycoprotein,lipoprotein, polysaccharide, any modified derivative of the abovemolecules, or any complex comprising one or more of the above molecules.Nucleic acids include DNA and RNA, can be single- or double-stranded;can be linear, branched or circular; and can be of any length. Nucleicacids include those capable of forming duplexes, as well astriplex-forming nucleic acids. See, for example, U.S. Pat. Nos.5,176,996 and 5,422,251. Proteins include, but are not limited to,DNA-binding proteins, transcription factors, chromatin remodelingfactors, methylated DNA binding proteins, polymerases, methylases,demethylases, acetylases, deacetylases, kinases, phosphatases,integrases, recombinases, ligases, topoisomerases, gyrases andhelicases.

An exogenous molecule can be the same type of molecule as an endogenousmolecule, e.g., an exogenous protein or nucleic acid. For example, anexogenous nucleic acid can comprise an infecting viral genome, a plasmidor episome introduced into a cell, or a chromosome that is not normallypresent in the cell. Methods for the introduction of exogenous moleculesinto cells are known to those of skill in the art and include, but arenot limited to, lipid-mediated transfer (i.e., liposomes, includingneutral and cationic lipids), electroporation, direct injection, cellfusion, particle bombardment, calcium phosphate co-precipitation,DEAE-dextran-mediated transfer and viral vector-mediated transfer.

By contrast, an “endogenous” molecule is one that is normally present ina particular cell at a particular developmental stage under particularenvironmental conditions. For example, an endogenous nucleic acid cancomprise a chromosome, the genome of a mitochondrion, chloroplast orother organelle, or a naturally-occurring episomal nucleic acid.Additional endogenous molecules can include proteins, for example,transcription factors and enzymes.

A “fusion” molecule is a molecule in which two or more subunit moleculesare linked, preferably covalently. The subunit molecules can be the samechemical type of molecule, or can be different chemical types ofmolecules. Examples of the first type of fusion molecule include, butare not limited to, fusion proteins (for example, a fusion between aTALE-repeat domain and a cleavage domain) and fusion nucleic acids (forexample, a nucleic acid encoding the fusion protein described supra).Examples of the second type of fusion molecule include, but are notlimited to, a fusion between a triplex-forming nucleic acid and apolypeptide, and a fusion between a minor groove binder and a nucleicacid.

Expression of a fusion protein in a cell can result from delivery of thefusion protein to the cell or by delivery of a polynucleotide encodingthe fusion protein to a cell, wherein the polynucleotide is transcribed,and the transcript is translated, to generate the fusion protein.Trans-splicing, polypeptide cleavage and polypeptide ligation can alsobe involved in expression of a protein in a cell. Methods forpolynucleotide and polypeptide delivery to cells are presented elsewherein this disclosure.

A “gene,” for the purposes of the present disclosure, includes a DNAregion encoding a gene product (see infra), as well as all DNA regionswhich regulate the production of the gene product, whether or not suchregulatory sequences are adjacent to coding and/or transcribedsequences. Accordingly, a gene includes, but is not necessarily limitedto, promoter sequences, terminators, translational regulatory sequencessuch as ribosome binding sites and internal ribosome entry sites,enhancers, silencers, insulators, boundary elements, replicationorigins, matrix attachment sites and locus control regions.

“Gene expression” refers to the conversion of the information, containedin a gene, into a gene product. A gene product can be the directtranscriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisenseRNA, ribozyme, structural RNA, shRNA, RNAi, miRNA or any other type ofRNA) or a protein produced by translation of a mRNA. Gene products alsoinclude RNAs which are modified, by processes such as capping,polyadenylation, methylation, and editing, and proteins modified by, forexample, methylation, acetylation, phosphorylation, ubiquitination,ADP-ribosylation, myristilation, and glycosylation.

A “gap size” refers to the nucleotides between the two TALE targetssites on the nucleic acid target. Gaps can be any size, including butnot limited to between 1 and 100 base pairs, or 5 and 30 base pairs,preferably between 10 and 25 base pairs, and more preferably between 12and 21 base pairs. Thus, a preferable gap size may be 12, 13, 14, 15,16, 17, 18, 19, 20, or 21 base pairs.

“Modulation” of gene expression refers to a change in the activity of agene. Modulation of expression can include, but is not limited to, geneactivation and gene repression. Genome editing (e.g., cleavage,alteration, inactivation, donor integration, random mutation) can beused to modulate expression. Gene inactivation refers to any reductionin gene expression as compared to a cell that does not include amodifier as described herein. Thus, gene inactivation may be partial orcomplete.

A “region of interest” is any region of cellular chromatin, such as, forexample, a gene or a non-coding sequence within or adjacent to a gene,in which it is desirable to bind an exogenous molecule. Binding can befor the purposes of targeted DNA cleavage and/or targeted recombination.A region of interest can be present in a chromosome, an episome, anorganellar genome (e.g., mitochondrial, chloroplast), or an infectingviral genome, for example. A region of interest can be within the codingregion of a gene, within transcribed non-coding regions such as, forexample, leader sequences, trailer sequences or introns, or withinnon-transcribed regions, either upstream or downstream of the codingregion. A region of interest can be as small as a single nucleotide pairor up to 2,000 nucleotide pairs in length, or any integral value ofnucleotide pairs.

The terms “operative linkage” and “operatively linked” (or “operablylinked”) are used interchangeably with reference to a juxtaposition oftwo or more components (such as sequence elements), in which thecomponents are arranged such that both components function normally andallow the possibility that at least one of the components can mediate afunction that is exerted upon at least one of the other components. Byway of illustration, a transcriptional regulatory sequence, such as apromoter, is operatively linked to a coding sequence if thetranscriptional regulatory sequence controls the level of transcriptionof the coding sequence in response to the presence or absence of one ormore transcriptional regulatory factors: A transcriptional regulatorysequence is generally operatively linked in cis with, a coding sequence,but need not be directly adjacent to it. For example, an enhancer is atranscriptional regulatory sequence that is operatively linked to acoding sequence, even though they are not contiguous.

With respect to fusion polypeptides, the term “operatively linked” canrefer to the fact that each of the components performs the same functionin linkage to the other component as it would if it were not so linked.For example, with respect to a fusion polypeptide in which a TALE-repeatdomain is fused to a cleavage domain, the TALE-repeat domain and thecleavage domain are in operative linkage if, in the fusion polypeptide,the TALE-repeat domain portion is able to bind its target site and/orits binding site, while the cleavage domain is able to cleave DNA in thevicinity of the target site.

A “functional fragment” of a protein, polypeptide or nucleic acid is aprotein, polypeptide or nucleic acid whose sequence is not identical tothe full-length protein, polypeptide or nucleic acid, yet retains thesame or has enhanced function as compared to the full-length protein,polypeptide or nucleic acid. Additionally, a functional fragment mayhave lesser function than the full-length protein, polypeptide ornucleic acid, but still have adequate function as defined by the user. Afunctional fragment can possess more, fewer, or the same number ofresidues as the corresponding native molecule, and/or can contain one ormore amino acid or nucleotide substitutions. Methods for determining thefunction of a nucleic acid (e.g., coding function, ability to hybridizeto another nucleic acid) are well-known in the art. Similarly, methodsfor determining protein function are well-known. For example, theDNA-binding function of a polypeptide can be determined, for example, byfilter-binding, electrophoretic mobility-shift, or immunoprecipitationassays. DNA cleavage can be assayed by gel electrophoresis. See Ausubelet al., supra. The ability of a protein to interact with another proteincan be determined, for example, by co-immunoprecipitation, two-hybridassays or complementation, both genetic and biochemical. See, forexample, Fields et al. (1989) Nature 340:245-246; U.S. Pat. No.5,585,245 and PCT WO 98/44350.

TALE-repeat domains can be “engineered” to bind to a predeterminednucleotide sequence, for example via engineering (altering one or moreamino acids) of the hypervariable diresidue region, for examplepositions 12 and/or 13 of a repeat unit within a TALE protein. In someembodiments, the amino acids at positions 4, 11, and 32 may beengineered. In other embodiments, atypical RVDs may be selected for usein an engineered TALE protein, enabling specification of a wider rangeof non-natural target sites. For example, a NK RVD may be selected foruse in recognizing a G nucleotide in the target sequence. In otherembodiments, amino acids in the repeat unit may be altered to change thecharacteristics (i.e. stability or secondary structure) of the repeatunit. Therefore, engineered TALE proteins are proteins that arenon-naturally occurring. In some embodiments, the genes encoding TALErepeat domains are engineered at the DNA level such that the codonsspecifying the TALE repeat amino acids are altered, but the specifiedamino acids are not (e.g., via known techniques of codon optimization).Non-limiting examples of engineered TALE proteins are those obtained bydesign and/or selection. A designed TALE protein is a protein notoccurring in nature whose design/composition results principally fromrational criteria. Rational criteria for design include application ofsubstitution rules and computerized algorithms for processinginformation in a database storing information of existing TALE designsand binding data. A “selected” TALE-repeat domain is a non-naturallyoccurring or atypical domain whose production results primarily from anempirical process such as phage display, interaction trap or hybridselection.

A “multimerization domain” is a domain incorporated at the amino,carboxy or amino and carboxy terminal regions of a TALE-fusion protein.These domains allow for multimerization of multiple TALE-fusion proteinunits. Examples of multimerization domains include leucine zippers.Multimerization domains may also be regulated by small molecules whereinthe multimerization domain assumes a proper conformation to allow forinteraction with another multimerization domain only in the presence ofa small molecule or external ligand. In this way, exogenous ligands canbe used to regulate the activity of these domains.

The target sites useful in the above methods can be subject toevaluation by other criteria or can be used directly for design orselection (if needed) and production of a TALE-fusion protein specificfor such a site. A further criterion for evaluating potential targetsites is their proximity to particular regions within a gene. Targetsites can be selected that do not necessarily include or overlapsegments of demonstrable biological significance with target genes, suchas regulatory sequences. Other criteria for further evaluating targetsegments include the prior availability of TALE-fusion proteins bindingto such segments or related segments, and/or ease of designing newTALE-fusion proteins to bind a given target segment.

After a target segment has been selected, a TALE-fusion protein thatbinds to the segment can be provided by a variety of approaches. Once aTALE-fusion protein has been selected, designed, or otherwise providedto a given target segment, the TALE-fusion protein or the DNA encodingit are synthesized. Exemplary methods for synthesizing and expressingDNA encoding TALE-repeat domain-containing proteins are described below.The TALE-fusion protein or a polynucleotide encoding it can then be usedfor modulation of expression, or analysis of the target gene containingthe target site to which the TALE-fusion protein binds.

TALE DNA Binding Domains

The polypeptides described herein comprise one or more (e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or evenmore) TALE-repeat units. TALE DNA binding domains, comprising multipleTALE-repeat units, have been studied to determine the sequencesresponsible for specificity. Within one organism, the TALE repeatstypically are highly conserved (except for the RVD) but may not be wellconserved across different species.

A TALE-repeat unit as found in the polypeptides described herein isgenerally of the form:X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-(X^(RVD))₂-(X)₂₀₋₂₂ (SEQ ID NO:399),where X is any amino acid and X^(RVD) (positions 12 and 13) involved inDNA binding. Non-limiting exemplary embodiments of such domains include:embodiments in which X¹ comprises a leucine (L), or methionine (M)residue; embodiments in which X¹⁰ comprises an alanine (A) residue or avaline (V) residue; embodiments in which (X)₂₀₋₂₂ comprises the sequence(Gly or Ser)-(X)₁₉₋₂₁ (SEQ ID NO:400); embodiments in which (X)₂₀₋₂₂comprises the sequence (X)₃₋₄-(Ala or Thr)-(X)₁₆₋₁₇ (SEQ ID NO:401);embodiments in which (X)₂₀₋₂₂ comprises the sequence (X)₄₋₅-(Leu orVal)-(X)₁₅₋₁₆ (SEQ ID NO:402); and combinations of any of the aboveembodiments (e.g., X′ comprises a leucine (L) or methionine (M) residueand X¹⁰ comprises an alanine (A) residue; X¹ comprises L or M and(X)₂₀₋₂₂ comprises the sequence Gly/Ser-(X)₁₉₋₂₁; (W)₂₀₋₂₂ comprises thesequence Gly/Ser-(X)₂₋₃-Ala/Thr-(X)₁₆₋₁₇; X¹⁰ comprises an alanine (A)or valine (V) residue and (X)₂₀₋₂₂ comprises the sequenceGly/Ser-(X)₁₉₋₂₁, etc.).

The TALE-repeat units of the compositions and methods described hereinmay be derived from any suitable TALE-protein. Non-limiting examples ofTALE proteins include TALE proteins derived from Ralstonia spp. orXanthamonas spp. Thus, in some embodiments, the DNA-binding domaincomprises one or more one or more naturally occurring and/or engineeredTALE-repeat units derived from the plant pathogen Xanthomonas (see Bochet al, (2009) Science 326: 1509-1512 and Moscou and Bogdanove, (2009)Science 326:1501). In other embodiments, the DNA-binding domaincomprises one or more naturally occurring and/or engineered TALE-repeatunits derived from the plant pathogen Ralstonia solanacearum, or otherTALE DNA binding domain from the TALE protein family. The TALE DNAbinding domains as described herein (comprising at least one TALE repeatunit) can include (i) one or more TALE repeat units not found in nature;(ii) one or more naturally occurring TALE repeat units; (iii) one ormore TALE repeat units with atypical RVDs; and combinations of (i), (ii)and/or (iii). In some embodiments, a TALE DNA binding domain of theinvention consists of completely non-naturally occurring or atypicalrepeat units. Furthermore, in polypeptides as described hereincomprising two or more TALE-repeat units, the TALE-repeat units(naturally occurring or engineered) may be derived from the same speciesor alternatively, may be derived from different species.

Table 1 shows an alignment of exemplary repeat units within two TALEproteins. Each TALE repeat is shown on a separate line with the columnsindicating the type of repeat, position of the start of that repeat, thename of the repeat, the residues at the hypervariable positions, and theentire repeat sequence.

TABLE 1 Comparison of TALE DNA binding domains from two TALEs fromXanthomonas Type Start Name RVD Repeat Sequence TALE AAA27592.1 (6.0repeats) full 288 R1.0 NI LTPEQVVAIASNIGGKQALETVQALLPVLCQAHG (SEQ ID NO:2) full 322 R2.0 NG LTPDQVVAIASNGGGKQALETVQRLLPVLCQAHG (SEQ ID NO: 3)full 356 R3.0 NI LTPEQVVAIASNIGGKQALETVQRLLPVLCQAHG (SEQ ID NO: 4) full390 R4.0 NI LTPEQVVAIASNIGGKQALETVQRLLPVLCQAHG (SEQ ID NO: 5) full 424R5.0 NG LTPEQVVAIASNGGGKQALETVQRLLPVLCQAHG (SEQ ID NO: 6) full 458 R6.0NG LTPEQVVAIASNGGGKQALETVQRLLPVLCQAHG (SEQ ID NO: 6) TALE AAA92974.1(15.5 repeats): full 287 R1.0 NI LTPDQVVAIASNIGGNQALETVQRLLPVLCQAHG (SEQID NO: 9) full 321 R2.0 HG LTPDQVVAIASHGGGKQALETVQRLLPVLCQAHG (SEQ IDNO: 10) full 355 R3.0 NI LTPDQVVAIASNIGGKQALATVQRLLPVLCQDHG (SEQ ID NO:11) full 389 R4.0 HG LTPDQVVAIASHGGGKQALETVQRLLPVLCQDHG (SEQ ID NO: 12)full 423 R5.0 NI LTPDQVVAIASNIGGKQALETVQRLLPVLCQDHG (SEQ ID NO: 13) full457 R6.0 NI LTPDQVVAIASNIGGKQALETVQRLLPVLCQDHG (SEQ ID NO: 14) full 491R7.0 NN LTPDQVVAIASNNGGKQALETVQRLLPVLCQTHG (SEQ ID NO: 15) full 525 R8.0HD LTPDQVVAIANHDGGKQALETVQRLLPVLCQDHG (SEQ ID NO: 16) full 559 R9.0 NILTPDQVVAIASNIGGKQALATVQRLLPVLCQAHG (SEQ ID NO: 17) full 593 R10.0 HDLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHG (SEQ ID NO: 18) full 627 R11.0 NNLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHG (SEQ ID NO: 19) full 661 R12.0 HGLTPAQVVAIANHGGGKQALETVQRLLPVLCQDHG (SEQ ID NO: 20) full 695 R13.0 NSLTPVQVVAIASNSGGKQALETVQRLLPVLCQDHG (SEQ ID NO: 21) full 729 R14.0 NGLTPVQVVAIASNGGGKQALATVQRLLPVLCQDHG (SEQ ID NO: 22) full 763 R15.0 HDLTPVQVVAIASHDGGKQALETVQRLLPVLCQDHG (SEQ ID NO: 23) half 797 R15.5 NGLTPDQVVAIASNGG-KQALESIVAQLSRPDPALAA (SEQ ID NO: 24)

Several TALE DNA binding proteins have been identified and can be foundin a standard GenBank search, including: AAB00675.1, (13.5 TALErepeats), AAB69865.1 (13.5 repeats), AAC43587.1 (17.5 repeats),AAD01494.1 (12.5 repeats), AAF98343.1 (25.5 repeats), AAG02079.2 (25.5repeats), AAN01357.1 (8.5 repeats), AAO72098 (17.5 repeats), AAQ79773.2(5.5 repeats), AAS46027.1 (28.5 repeats), AAS58127.2 (13.5 repeats),AAS58128.2 (17.5 repeats), AAS58129.3 (18.5 repeats), AAS58130.3 (9.5repeats), AAT46123.1 (22.5 repeats), AAT46124.1 (26.5 repeats),AAW59491.1 (5.5 repeats), AAW59492.1 (16.5 repeats), AAW59493.1 (19.5repeats), AAW77510.1 (5.5 repeats), AAY43358 (21.5 repeats), AAY43359.1(11.5 repeats), AAY43360.1 (14.5 repeats), AAY54166.1 (19.5 repeats),AAY54168.1 (16.5 repeats), AAY54169.1 (12.5 repeats), AAY54170.1 (23.5repeats), ABB70129.1 (21.5 repeats), ABB70183.1 (22.5 repeats),ABO77779.1 (17.5 repeats), etc.

TALE type proteins have also been found in the bacterium Ralstoniasolanacearum and Table 2 lists a similar comparison of two examples ofthese DNA binding domains:

TABLE 2 Comparison of TALE DNA binding domains from two TALE fromRalstonia Type Start Name RVD Repeat Sequence TALE ABO27067.1 (13.5repeats) full 0 R1.0 NT LTPQQVVAIASNTGGKRALEAVCVQLPVLRAAPYR (SEQ ID NO:25) full 35 R2.0 NK LSTEQVVAIASNKGGKQALEAVKAHLLDLLGAPYV (SEQ ID NO: 26)full 70 R3.0 HN LDTEQVVAIASHNGGKQALEAVKADLLDLRGAPYA (SEQ ID NO: 27) full105 R4.0 HN LSTEQVVAIASHNGGKQALEAVKADLLDLRGAPYA (SEQ ID NO: 28) full 140R5.0 HN LSTEQVVAIASHNGGKQALEAVKAQLLDLRGAPYA (SEQ ID NO: 29) full 175R6.0 HN LSTAQVVAIASHNGGKQALEAVKAQLLDLRGAPYA (SEQ ID NO: 30) full 210R7.0 NG LSTAQVVAIASNGGGKQALEGIGEQLLKLRTAPYG (SEQ ID NO: 31) full 245R8.0 SH LSTEQVVAIASSHGGKQALEAVRALFPDLRAAPYA (SEQ ID NO: 32) full 280R9.0 NP LSTAQLVAIASNPGGKQALEAVRALFRELRAAPYA (SEQ ID NO: 33) full 315R10.0 NH LSTEQVVAIASNHGGKQALEAVRALFRELRAAPYA (SEQ ID NO: 34) full 350R11.0 NH LSTEQVVAIASNHGGKQALEAVRALFRGLRAAPYG (SEQ ID NO: 35) full 385R12.0 SN LSTAQVVAIASSNGGKQALEAVWALLPVLRATPYD (SEQ ID NO: 36) full 420R13.0 HY LNTAQVVAIASHYGGKPALEAVWAKLPVLRGVPYA (SEQ ID NO: 37) half 455R13.5 IS LSTAQVVAIACISG-QQALEAIEAHMPTLRQAPH (SEQ ID NO: 38) TALEABO27068.1 (4.5 repeats) full 0 R1.0 NPLSTAQLVAIASNPGGKQALEAVRAPFREVRAAPYA (SEQ ID NO: 39) full 35 R2.0 NHLSPEQVVAIASNHGGKQALEAVRALFRGLRAAPYG (SEQ ID NO: 40) full 70 R3.0 SNLSTAQVVAIASSNGGKQALEAVWALLPVLRATPYD (SEQ ID NO: 41) full 105 R4.0 HDLSTAQVVAIASHDGGKPALEAVWAKLPVLRGAPYA (SEQ ID NO: 42) half 140 R4.5 ISLSTAQVVAIACISG-QQALEAIEAHMPTLRQAPH (SEQ ID NO: 43)

Additional examples of TALE type proteins from Ralstonia includeABO27069.1 (10.5 repeats), ABO27070.1 (11.5 repeats), ABO27071.1 (7.5repeats), ABO27072.1 (3.5 repeats), etc.

The DNA-binding polypeptides comprising TALE-repeat domains as describedherein may also include additional TALE polypeptide sequences, forexample N-terminal (N-cap) sequences and, optionally, C-terminal (C-cap)sequences flanking the repeat domains. N-cap sequences may be naturallyor non-naturally occurring sequences of any length sufficient to supportthe function (e.g., DNA-binding, cleavage, activation, etc.) of theDNA-binding polypeptide and fusion proteins comprising these TALE-repeatdomain-containing DNA-binding polypeptides. In certain embodiments, theprotein comprises an N-cap sequence comprising a fragment (truncation)of a region of a TALE protein N-terminal to the repeat domain (e.g., anN-cap sequence comprising at least 130 to 140 residues (e.g., 131, 132,133, 134, 135, 136, 137, 138, 139 or 140 residues) of a TALE polypeptideN-terminal of the repeat domain). In other embodiments, the TALE-repeatdomain polypeptides as described herein the protein comprises a C-capsequence comprising a fragment (truncated) region of a TALE proteinC-terminal to the repeat domain (e.g., an C-cap sequence comprising C−20to C+28; C−20 to C+55, or C−20 to C+63). In certain embodiments, theC-cap sequence comprises a half-repeat (C−20 to C−1). The TALEDNA-binding polypeptides as described herein may include N-cap, C-capsequences or both N-cap and C-cap sequences.

The complete protein sequences (including TALE repeat domains as well asN-terminal and C-terminal sequences) of the TALE repeats shown in Table1 and 2 are shown below in Table 3. The TALE repeat sequences of Tables1 and 2 are shown in bold.

TABLE 3 complete amino acid sequence for GenBank accession numbersAAA27592.1, AAA92974.1, ABO27067.1 and ABO27068.1. AAA27592.1 (SEQ IDNO: 44) MDPIRSRTPSPARELLPGPQPDGVQPTADRGVSPPAGGPLDGLPARRTMSRTRLPSPPAPSPAFSAGSFSDLLRQFDPSLFNTSLFDSLPPFGAHHTEAATGEWDEVQSGLRAADAPPPTMRVAVTAARPPRAKPAPRRRAAQPSDASPAAQVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLI AAA92974.1 (SEQ ID NO: 45)MDPIRSRTPSPARELLPGPQPDRVQPTADRGGAPPAGGPLDGLPARRTMSRTRLPSPPAPSPAFSAGSFSDLLRQFDPSLLDTSLLDSMPAVGTPHTAAAPAECDEVQSGLRAADDPPPTVRVAVTARPPRAKPAPRRRAAQPSDASPAAQVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVTYQDIIRALPEATHEDIVGVGKQWSGARALEALLTEAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNIGGNQALETVQRLLPVLCQAHGLTPDQVVAIASHGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALATVQRLLPVLCQDHGLTPDQVVAIASHGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQTHGLTPDQVVAIANHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALATVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIANHGGGKQALETVQRLLPVLCQDHGLTPVQVVAIASNSGGKQALETVQRLLPVLCQDHGLTPVQVVAIASNGGGKQALATVQRLLPVLCQDHGLTPVQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPELIRRINRRIPERTSHRVADLAHVVRVLGFFQSHSHPAQAFDDAMTQFGMSRHGLAQLFRRVGVTELEARYGTLPPASQRWDRILQASGMKRVKPSPTSAQTPDQASLHAFADSLERDLDAPSPMHEGDQTRASSRKRSRSDRAVTGPSTQQSFEVRVPEQQDALHLPLSWRVKRPRTRIGGGLPDPGTPIAADLAASSTVMWEQDAAPFAGAADDFPAFNEEELAWLMELLPQSGSVGGTI ABO27068.1 (SEQ ID NO: 46)LSTAQLVAIASNPGGKQALEAVRAPFREVRAAPYALSPEQVVAIASNHGGKQALEAVRALFRGLRAAPYGLSTAQVVAIASSNGGKQALEAVWALLPVLRATPYDLSTAQVVAIASHDGGKPALEAVWAKLPVLRGAPYALSTAQVVAIACISGQQALEAIEAH MPTLRQAPHSABO27067.1 (SEQ ID NO: 47)LTPQQVVAIASNTGGKRALEAVCVQLPVLRAAPYRLSTEQVVAIASNKGGKQALEAVKAHLLDLLGAPYVLDTEQVVAIASHNGGKQALEAVKADLLDLRGAPYALSTEQVVAIASHNGGKQALEAVKADLLDLRGAPYALSTEQVVAIASHNGGKQALEAVKAQLLDLRGAPYALSTAQVVAIASHNGGKQALEAVKAQLLDLRGAPYALSTAQVVAIASNGGGKQALEGIGEQLLKLRTAPYG - [unknown sequence] -LSTEQVVAIASSHGGKQALEAVRALFPDLRAAPYALSTAQLVAIASNPGGKQALEAVRALFRELRAAPYALSTEQVVAIASNHGGKQALEAVRALFRELRAAPYALSTEQVVAIASNHGGKQALEAVRALFRGLRAAPYGLSTAQVVAIASSNGGKQALEAVWALLPVLRATPYDLNTAQVVAIASHYGGKPALEAVWAKLPVLRGVPYALSTAQVVAIACISGQQALEAIEAHMPTLRQAPHGLSPERVAAIACIGGRSAVEA

Artificial TALE proteins and TALE fusion proteins can be produced tobind to a novel sequence using natural or engineered TALE repeat units(see Boch et al, ibid and Morbitzer et al, (2010) Proc. Natl. Acad. Sci.USA 107(50):21617-21622). See, also e.g., WO 2010/079430. When thisnovel target sequence was inserted upstream of a reporter gene in plantcells, the researchers were able to demonstrate activation of thereporter gene. Artificial TALE fusions comprising the FokI cleavagedomain can also cleave DNA in living cells (see Christin et al, ibid, Liet al (2011a) and (2011b) ibid, Cernak et al (2011) Nucl. Acid. Res.epub doi:10.1093/nar/gcr218.

An engineered TALE protein and TALE fusion protein can have a novelbinding specificity, compared to a naturally-occurring TALE protein.Engineering methods include, but are not limited to, rational design andvarious types of selection. Rational design includes, for example, usingdatabases comprising nucleotide sequences for modules for single ormultiple TALE repeats. Exemplary selection methods, including phagedisplay and two-hybrid systems, are disclosed in U.S. Pat. Nos.5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466;6,200,759; and 6,242,568; as well as WO 98/37186; WO 98/53057; WO00/27878; WO 01/88197 and GB 2,338,237. In naturally occurring TALEproteins, only a limited repertoire of potential dipeptide motifs aretypically employed. Thus, as described herein, TALE related domainscontaining all possible mono- and di-peptide sequences have beenconstructed and assembled into candidate TALE proteins. Thus, in certainembodiments, one or more TALE-repeat units of the DNA-binding proteincomprise atypical RVDs.

Additionally, in naturally occurring TALE proteins of the same species,the repeat units often show little variability within the frameworksequence (i.e. the residue(s) not involved in direct DNA contact(non-RVD residues). This lack of variability may be due to a number offactors including evolutionary relationships between individual TALErepeat units and protein folding requirements between adjacent repeats.Between differing phytopathogenicbacterial species however the frameworksequences can vary. For example, the TALE repeat sequences in theXanthomonas campestris pv vesicatoria, the protein AvrBs3 has less than40% homology with brg11 and hpx17 repeat units from Ralstoniasolanacearum (see Heuer et al (2007) Appl Environ Micro 73 (13):4379-4384). The TALE repeat may be under stringent functional selectionin each bacterium's natural environment, e.g., from the sequence of thegenes in the host plant that the TALE regulates. Thus, as describedherein, variants in the TALE framework (e.g., within the TALE repeatunit or sequences outside the repeat units such as N-cap and C-capsequences) may be introduced by targeted or random mutagenesis byvarious methods know in the art, and the resultant TALE fusion proteinsscreened for optimal activity.

Multi TALE repeat modules may also be useful not only for assembling theDNA binding domains (comprising at least one TALE repeat unit) asdescribed above, but also may be useful for the assembly of mini-TALEmultimers (i.e. trimers, tetramers, pentamers etc.), wherein spanninglinkers that also functioned as capping regions between the mini-TALEDNA binding domains would allow for base skipping and may result inhigher DNA binding specificity. The use of linked mini-TALE DNA bindingdomains would relax the requirement for strict functional modularity atthe level of individual TALE repeats and allows for the development ofmore complex and/or specific DNA recognition schemes wherein amino acidsfrom adjacent motifs within a given module might be free to interactwith each other for cooperative recognition of a desired DNA targetsequence. Mini-TALE DNA binding domains could be linked and expressedusing a suitable selection system (i.e. phage display) with randomizeddipeptide motifs (or any other identified key positions) and selectedbased on their nucleic acid binding characteristics. Alternatively,multi-TALE repeat modules may be used to create an archive of repeatmodules to allow for rapid construction of any specific desiredTALE-fusion protein.

Selection of target sites and methods for design and construction offusion proteins (and polynucleotides encoding same) are known to thoseof skill in the art and described in detail in U.S. Patent ApplicationPublication Nos. 20050064474 and 20060188987, incorporated by referencein their entireties herein.

Artificial fusion proteins linking TALE DNA binding domains to zincfinger DNA binding domains may also be produced. These fusions may alsobe further linked to a desired functional domain.

In addition, as disclosed in these and other references, TALE DNAbinding domains and/or zinc finger domains may be linked together usingany suitable linker sequences, including for example, linkers of 5 ormore amino acids in length (e.g., TGEKP (SEQ ID NO:48), TGGQRP (SEQ IDNO:49), TGQKP (SEQ ID NO:50), and/or TGSQKP (SEQ ID NO:51)), although itis likely that sequences that can function as capping sequence (N-capand C-cap sequences) would be required at the interface between the TALErepeat domain and the linker. Thus, when linkers are used, linkers offive or more amino acids can be used in conjunction with the capsequences to join the TALE DNA binding domains to a desired fusionpartner domain. See, also, U.S. Pat. Nos. 6,479,626; 6,903,185; and7,153,949 for exemplary linker sequences 6 or more amino acids inlength. In addition, linkers between the TALE repeat domains and thefused functional protein domains can be constructed to be eitherflexible or positionally constrained to allow for the most efficientgenomic modification. Linkers of varying lengths and compositions may betested.

Fusion Proteins

Fusion proteins comprising DNA-binding proteins (e.g., TALE-fusionproteins) as described herein and a heterologous regulatory orfunctional domain (or functional fragment thereof) are also provided.Common domains include, e.g., transcription factor domains (activators,repressors, co-activators, co-repressors), nuclease domains, silencerdomains, oncogene domains (e.g., myc, jun, fos, myb, max, mad, rel, ets,bcl, myb, mos family members etc.); DNA repair enzymes and theirassociated factors and modifiers; DNA rearrangement enzymes and theirassociated factors and modifiers; chromatin associated proteins andtheir modifiers (e.g. kinases, acetylases and deacetylases); and DNAmodifying enzymes (e.g., methyltransferases, topoisomerases, helicases,ligases, kinases, phosphatases, polymerases, endonucleases), DNAtargeting enzymes such as transposons, integrases, recombinases andresolvases and their associated factors and modifiers, nuclear hormonereceptors, nucleases (cleavage domains or half-domains) and ligandbinding domains. Other fusion proteins may include reporter or selectionmarkers. Examples of reporter domains include GFP, GUS and the like.Reporters with specific utility in plant cells include GUS.

Suitable domains for achieving activation include the HSV VP16activation domain (see, e.g., Hagmann et al., J. Virol. 71, 5952-5962(1997)) nuclear hormone receptors (see, e.g., Torchia et al., Curr.Opin. Cell. Biol. 10:373-383 (1998)); the p65 subunit of nuclear factorkappa B (Bitko & Barik, J. Virol. 72:5610-5618 (1998) and Doyle & Hunt,Neuroreport 8:2937-2942 (1997)); Liu et al., Cancer Gene Ther. 5:3-28(1998)), or artificial chimeric functional domains such as VP64 (Beerliet al., (1998) Proc. Natl. Acad. Sci. USA 95:14623-33), and degron(Molinari et al., (1999) EMBO J. 18, 6439-6447). Additional exemplaryactivation domains include, Oct 1, Oct-2A, Sp1, AP-2, and CTF1 (Seipelet al., EMBO J. 11, 4961-4968 (1992) as well as p300, CBP, PCAF, SRC1PvALF, AtHD2A and ERF-2. See, for example, Robyr et al. (2000) Mol.Endocrinol. 14:329-347; Collingwood et al. (1999) J. Mol. Endocrinol.23:255-275; Leo et al. (2000) Gene 245:1-11; Manteuffel-Cymborowska(1999) Acta Biochim. Pol. 46:77-89; McKenna et al. (1999) J. SteroidBiochem. Mol. Biol. 69:3-12; Malik et al. (2000) Trends Biochem. Sci.25:277-283; and Lemon et al. (1999) Curr. Opin. Genet. Dev. 9:499-504.Additional exemplary activation domains include, but are not limited to,OsGAI, HALF-1, C1, AP1, ARF-5,-6,-7, and -8, CPRF1, CPRF4, MYC-RP/GP,and TRAB1. See, for example, Ogawa et al. (2000) Gene 245:21-29; Okanamiet al. (1996) Genes Cells 1:87-99; Goff et al. (1991) Genes Dev.5:298-309; Cho et al. (1999) Plant Mol. Biol. 40:419-429; Ulmason et al.(1999) Proc. Natl. Acad. Sci. USA 96:5844-5849; Sprenger-Haussels et al.(2000) Plant J. 22:1-8; Gong et al. (1999) Plant Mol. Biol. 41:33-44;and Hobo et al. (1999) Proc. Natl. Acad. Sci. USA 96:15,348-15,353.

It will be clear to those of skill in the art that, in the formation ofa fusion protein (or a nucleic acid encoding same) between a DNA-bindingdomain as described herein and a functional domain, either an activationdomain or a molecule that interacts with an activation domain issuitable as a functional domain. Essentially any molecule capable ofrecruiting an activating complex and/or activating activity (such as,for example, histone acetylation) to the target gene is useful as anactivating domain of a fusion protein. Insulator domains, localizationdomains, and chromatin remodeling proteins such as ISWI-containingdomains and/or methyl binding domain proteins suitable for use asfunctional domains in fusion molecules are described, for example, inco-owned U.S. Patent Applications 2002/0115215 and 2003/0082552 and inco-owned WO 02/44376.

Exemplary repression domains include, but are not limited to, KRAB A/B,KOX, TGF-beta-inducible early gene (TIEG), v-erbA, SID, MBD2, MBD3,members of the DNMT family (e.g., DNMT1, DNMT3A, DNMT3B), Rb, and MeCP2.See, for example, Bird et al. (1999) Cell 99:451-454; Tyler et al.(1999) Cell 99:443-446; Knoepfler et al. (1999) Cell 99:447-450; andRobertson et al. (2000) Nature Genet. 25:338-342. Additional exemplaryrepression domains include, but are not limited to, ROM2 and AtHD2A.See, for example, Chem et al. (1996) Plant Cell 8:305-321; and Wu et al.(2000) Plant J. 22:19-27.

In certain embodiments, the target site bound by the TALE-fusion proteinis present in an accessible region of cellular chromatin. Accessibleregions can be determined as described, for example, in co-ownedInternational Publication WO 01/83732. If the target site is not presentin an accessible region of cellular chromatin, one or more accessibleregions can be generated as described in co-owned WO 01/83793. Inadditional embodiments, the DNA-binding domain of a fusion molecule iscapable of binding to cellular chromatin regardless of whether itstarget site is in an accessible region or not. For example, suchDNA-binding domains are capable of binding to linker DNA and/ornucleosomal DNA. Examples of this type of “pioneer” DNA binding domainare found in certain steroid receptor and in hepatocyte nuclear factor 3(HNF3). Cordingley et al. (1987) Cell 48:261-270; Pina et al. (1990)Cell 60:719-731; and Cirillo et al. (1998) EMBO J. 17:244-254.

The fusion molecule may be formulated with a pharmaceutically acceptablecarrier, as is known to those of skill in the art. See, for example,Remington's Pharmaceutical Sciences, 17th ed., 1985; and co-owned WO00/42219.

The functional component/domain of a fusion molecule can be selectedfrom any of a variety of different components capable of influencingtranscription of a gene once the fusion molecule binds to a targetsequence via its DNA binding domain. Hence, the functional component caninclude, but is not limited to, various transcription factor domains,such as activators, repressors, co-activators, co-repressors, andsilencers.

Additional exemplary functional domains are disclosed, for example, inco-owned U.S. Pat. No. 6,534,261 and US Patent Application PublicationNo. 2002/0160940.

Functional domains that are regulated by exogenous small molecules orligands may also be selected. For example, RheoSwitch® technology may beemployed wherein a functional domain only assumes its activeconformation in the presence of the external RheoChem™ ligand (see forexample US 20090136465). Thus, the TALE-fusion protein may be operablylinked to the regulatable functional domain wherein the resultantactivity of the TALE-fusion protein is controlled by the externalligand.

In certain embodiments, the TALE DNA-binding proteins, or fragmentsthereof, are used as nucleases via fusion (N- and/or C-terminal to theTALE-repeat domain, N-cap and/or C-cap sequences) of a TALE DNA-bindingdomain to at least one nuclease (cleavage domain, cleavage half-domain).The cleavage domain portion of the fusion proteins disclosed herein canbe obtained from any endonuclease or exonuclease. Exemplaryendonucleases from which a cleavage domain can be derived include, butare not limited to, restriction endonucleases and homing endonucleases.See, for example, 2002-2003 Catalogue, New England Biolabs, Beverly, MA; and Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388. Additionalenzymes which cleave DNA are known (e.g., S1 Nuclease; mung beannuclease; pancreatic DNase I; micrococcal nuclease; yeast HOendonuclease; see also Linn et al. (eds.) Nucleases, Cold Spring HarborLaboratory Press, 1993). One or more of these enzymes (or functionalfragments thereof) can be used as a source of cleavage domains andcleavage half-domains.

Similarly, a cleavage half-domain can be derived from any nuclease orportion thereof, as set forth above, that requires dimerization forcleavage activity. In general, two fusion proteins are required forcleavage if the fusion proteins comprise cleavage half-domains.Alternatively, a single protein comprising two cleavage half-domains canbe used. The two cleavage half-domains can be derived from the sameendonuclease (or functional fragments thereof), or each cleavagehalf-domain can be derived from a different endonuclease (or functionalfragments thereof). In addition, the target sites for the two fusionproteins are preferably disposed, with respect to each other, such thatbinding of the two fusion proteins to their respective target sitesplaces the cleavage half-domains in a spatial orientation to each otherthat allows the cleavage half-domains to form a functional cleavagedomain, e.g., by dimerizing. Thus, in certain embodiments, the nearedges of the target sites are separated by 5-8 nucleotides or by 15-18nucleotides. However any integral number of nucleotides or nucleotidepairs can intervene between two target sites (e.g., from 2 to 50nucleotide pairs or more). In general, the site of cleavage lies betweenthe target sites.

Restriction endonucleases (restriction enzymes) are present in manyspecies and are capable of sequence-specific binding to DNA (at arecognition site), and cleaving DNA at or near the site of binding.Certain restriction enzymes (e.g., Type IIS) cleave DNA at sites removedfrom the recognition site and have separable binding and cleavagedomains. For example, the Type IIS enzyme Fok I catalyzesdouble-stranded cleavage of DNA, at 9 nucleotides from its recognitionsite on one strand and 13 nucleotides from its recognition site on theother. See, for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and5,487,994; as well as Li et al. (1992) Proc. Natl. Acad. Sci. USA89:4275-4279; Li et al. (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768;Kim et al. (1994a) Proc. Natl. Acad. Sci. USA 91:883-887; Kim et al.(1994b) J. Biol. Chem. 269:31,978-31,982. Thus, in one embodiment,fusion proteins comprise the cleavage domain (or cleavage half-domain)from at least one Type IIS restriction enzyme and one or more TALEDNA-binding domains, which may or may not be engineered.

Exemplary Type IIS restriction enzymes, whose cleavage domains areseparable from the binding domain, include Fok I and BfiI (see Zarembaet al, (2004) J Mol Biol. 336(1):81-92). Fok enzyme is active as a dimer(see Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95:10,570-10,575). For targeted double-stranded cleavage and/or targetedreplacement of cellular sequences using TALE repeat domain-Fok I fusions(or variants thereof further comprising a C-cap and an N-cap), twofusion proteins, each comprising a FokI cleavage half-domain, can beused to reconstitute a catalytically active cleavage domain.Alternatively, a single polypeptide molecule containing a TALE-repeatdomain and two Fok I cleavage half-domains can also be used. Anotherpreferred Type IIS restriction enzyme is BfiI (see Zaremba et al, (2004)J Mol Biol. 336(1):81-92). The cleavage domain of this enzyme may beseparated from its DNA binding domain and operably linked to a TALE DNAbinding domain to create a TALEN.

A cleavage domain or cleavage half-domain can be any portion of aprotein that retains cleavage activity, or that retains the ability tomultimerize (e.g., dimerize) to form a functional cleavage domain.

Exemplary Type IIS restriction enzymes are described in InternationalPublication WO 07/014,275, incorporated herein in its entirety.Additional restriction enzymes also contain separable binding andcleavage domains, and these are contemplated by the present disclosure.See, for example, Roberts et al. (2003) Nucleic Acids Res. 31:418-420.

To enhance cleavage specificity, in certain embodiments, the cleavagedomain comprises one or more engineered cleavage half-domain (alsoreferred to as dimerization domain mutants) that minimize or preventhomodimerization, as described, for example, in U.S. Patent PublicationNos. 20050064474; 20060188987, 20080131962, 20090311787; 20090305346;20110014616, and U.S. patent application Ser. No. 12/931,660, thedisclosures of all of which are incorporated by reference in theirentireties herein. Amino acid residues at positions 446, 447, 479, 483,484, 486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537, and 538 ofFok I are all targets for influencing dimerization of the Fok I cleavagehalf-domains.

Exemplary engineered cleavage half-domains of Fok I that form obligateheterodimers include a pair in which a first cleavage half-domainincludes mutations at amino acid residues at positions 490 and 538 ofFok I and a second cleavage half-domain includes mutations at amino acidresidues 486 and 499.

Additional engineered cleavage half-domains of Fok I form an obligateheterodimers can also be used in the fusion proteins described herein.The first cleavage half-domain includes mutations at amino acid residuesat positions 490 and 538 of Fok I and the second cleavage half-domainincludes mutations at amino acid residues 486 and 499.

Thus, in one embodiment, a mutation at 490 replaces Glu (E) with Lys(K); the mutation at 538 replaces Iso (I) with Lys (K); the mutation at486 replaced Gln (Q) with Glu (E); and the mutation at position 499replaces Iso (I) with Lys (K). Specifically, the engineered cleavagehalf-domains described herein were prepared by mutating positions 490(E→K) and 538 (I→K) in one cleavage half-domain to produce an engineeredcleavage half-domain designated “E490K:I538K” and by mutating positions486 (Q→E) and 499 (I→L) in another cleavage half-domain to produce anengineered cleavage half-domain designated “Q486E:I499L”. The engineeredcleavage half-domains described herein are obligate heterodimer mutantsin which aberrant cleavage is minimized or abolished. See, e.g., Example1 of U.S. Patent Publication No. 2008/0131962, the disclosure of whichis incorporated by reference in its entirety for all purposes.

The engineered cleavage half-domains described herein are obligateheterodimer mutants in which aberrant cleavage is minimized orabolished. See, e.g., Example 1 of WO 07/139,898. In certainembodiments, the engineered cleavage half-domain comprises mutations atpositions 486, 499 and 496 (numbered relative to wild-type FokI), forinstance mutations that replace the wild type Gln (Q) residue atposition 486 with a Glu (E) residue, the wild type Iso (I) residue atposition 499 with a Leu (L) residue and the wild-type Asn (N) residue atposition 496 with an Asp (D) or Glu (E) residue (also referred to as a“ELD” and “ELE” domains, respectively). In other embodiments, theengineered cleavage half-domain comprises mutations at positions 490,538 and 537 (numbered relative to wild-type FokI), for instancemutations that replace the wild type Glu (E) residue at position 490with a Lys (K) residue, the wild type Iso (I) residue at position 538with a Lys (K) residue, and the wild-type His (H) residue at position537 with a Lys (K) residue or a Arg (R) residue (also referred to as“KKK” and “KKR” domains, respectively). In other embodiments, theengineered cleavage half-domain comprises mutations at positions 490 and537 (numbered relative to wild-type FokI), for instance mutations thatreplace the wild type Glu (E) residue at position 490 with a Lys (K)residue and the wild-type His (H) residue at position 537 with a Lys (K)residue or a Arg (R) residue (also referred to as “KIK” and “KIR”domains, respectively). (See U.S. provisional application 61/337,769filed Feb. 8, 2010 and U.S. provisional application 61/403,916, filedSep. 23, 2010). In addition, the Fold nuclease domain variants includingmutations known as “Sharkey” or “Sharkey′ (Sharkey prime)” mutations maybe used (see Guo et al, (2010) J. Mol. Biol.doi:10.1016/j.jmb.2010.04.060).

Engineered cleavage half-domains described herein can be prepared usingany suitable method, for example, by site-directed mutagenesis ofwild-type cleavage half-domains (Fok I) as described in U.S. PatentPublication Nos. 20050064474, 20070134796; 20080131962.

TALE-fusion polypeptides and nucleic acids can be made using routinetechniques in the field of recombinant genetics. Basic texts disclosingthe general methods of use in this invention include Sambrook et al.,Molecular Cloning, A Laboratory Manual (2nd ed. 1989); Kriegler, GeneTransfer and Expression: A Laboratory Manual (1990); and CurrentProtocols in Molecular Biology (Ausubel et al., eds., 1994)). Inaddition, essentially any nucleic acid can be custom ordered from any ofa variety of commercial sources. Similarly, peptides and antibodies canbe custom ordered from any of a variety of commercial sources.

Two alternative methods are typically used to create the codingsequences required to express newly designed DNA-binding peptides. Oneprotocol is a PCR-based assembly procedure that utilizes overlappingoligonucleotides. These oligonucleotides contain substitutionsprimarily, but not limited to, positions 12 and 13 on the repeateddomains making them specific for each of the different DNA-bindingdomains. Additionally, amino acid substitutions may be made at positions4, 11 and 32. Amino acid substitutions may also be made at positions 2,3, 4, 21, 23, 24, 25, 27, 30, 31, 33, 34 and/or 35 within one repeatunit. In some embodiments, the repeat unit contains a substitution inone position, and in others, the repeat unit contains from 2 to 18 aminoacid substitutions. In some embodiments, the nucleotide sequence of therepeat units may be altered without altering the amino acid sequence.

Any suitable method of protein purification known to those of skill inthe art can be used to purify TALE-fusion proteins of the invention (seeAusubel, supra, Sambrook, supra). In addition, any suitable host can beused, e.g., bacterial cells, insect cells, yeast cells, mammalian cells,and the like.

Thus, fusion molecules are constructed by methods of cloning andbiochemical conjugation that are well known to those of skill in theart. Fusion molecules comprise a DNA-binding domain and a functionaldomain (e.g., a transcriptional activation or repression domain). Fusionmolecules also optionally comprise nuclear localization signals (suchas, for example, that from the SV40 medium T-antigen) and epitope tags(such as, for example, FLAG and hemagglutinin). Fusion proteins (andnucleic acids encoding them) are designed such that the translationalreading frame is preserved among the components of the fusion. Thefusion proteins as described herein may include one or more functionaldomains at the N- and/or C-terminus of the DNA-binding polypeptides asdescribed herein.

Fusions between a polypeptide component of a functional domain (or afunctional fragment thereof) on the one hand, and a non-proteinDNA-binding domain (e.g., antibiotic, intercalator, minor groove binder,nucleic acid) on the other, are constructed by methods of biochemicalconjugation known to those of skill in the art. See, for example, thePierce Chemical Company (Rockford, Ill.) Catalogue. Methods andcompositions for making fusions between a minor groove binder and apolypeptide have been described. Mapp et al. (2000) Proc. Natl. Acad.Sci. USA 97:3930-3935.

Additional Methods for Targeted Cleavage

Any nuclease having a target site in any desired gene(s) can be used inthe methods disclosed herein. For example, homing endonucleases andmeganucleases have very long recognition sequences, some of which arelikely to be present, on a statistical basis, once in a human-sizedgenome. Any such nuclease having a target site in a desired gene can beused instead of, or in addition to, a TALE-repeat domain nucleasefusion, including for example, a zinc finger nuclease and/or ameganuclease, for targeted cleavage.

In certain embodiments, the nuclease is a meganuclease (homingendonuclease). Naturally-occurring meganucleases recognize 15-40base-pair cleavage sites and are commonly grouped into four families:the LAGLIDADG family, the GIY-YIG family, the His-Cyst box family andthe HNH family. Exemplary homing endonucleases include I-SceI, I-CeuI,PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII,I-CreI, I-TevI, I-TevII and I-TevIII. Their recognition sequences areknown. See also U.S. Pat. No. 5,420,032; U.S. Pat. No. 6,833,252;Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388; Dujon et al.(1989) Gene 82:115-118; Perler et al. (1994) Nucleic Acids Res. 22,1125-1127; Jasin (1996) Trends Genet. 12:224-228; Gimble et al. (1996)J. Mol. Biol. 263:163-180; Argast et al. (1998) J. Mol. Biol.280:345-353 and the New England Biolabs catalogue.

DNA-binding domains from naturally-occurring meganucleases, primarilyfrom the LAGLIDADG family, have been used to promote site-specificgenome modification in plants, yeast, Drosophila, mammalian cells andmice, but this approach has been limited to the modification of eitherhomologous genes that conserve the meganuclease recognition sequence(Monet et al. (1999), Biochem. Biophysics. Res. Common. 255: 88-93) orto pre-engineered genomes into which a recognition sequence has beenintroduced (Route et al. (1994), Mol. Cell. Biol. 14: 8096-106; Chiltonet al. (2003), Plant Physiology. 133: 956-65; Puchta et al. (1996),Proc. Natl. Acad. Sci. USA 93: 5055-60; Rong et al. (2002), Genes Dev.16: 1568-81; Gouble et al. (2006), J. Gene Med. 8(5):616-622).Accordingly, attempts have been made to engineer meganucleases toexhibit novel binding specificity at medically or biotechnologicallyrelevant sites (Porteus et al. (2005), Nat. Biotechnol. 23: 967-73;Sussman et al. (2004), J. Mol. Biol. 342: 31-41; Epinat et al. (2003),Nucleic Acids Res. 31: 2952-62; Chevalier et al. (2002) Molec. Cell10:895-905; Epinat et al. (2003) Nucleic Acids Res. 31:2952-2962;Ashworth et al. (2006) Nature 441:656-659; Paques et al. (2007) CurrentGene Therapy 7:49-66; U.S. Patent Publication Nos. 20070117128;20060206949; 20060153826; 20060078552; and 20040002092).

Delivery

The TALE-fusion proteins, polynucleotides encoding same and compositionscomprising the proteins and/or polynucleotides described herein may bedelivered to a target cell by any suitable means, including, forexample, by injection of mRNA encoding the TAL-fusion protein. See,Hammerschmidt et al. (1999) Methods Cell Biol. 59:87-115.

Methods of delivering proteins comprising engineered transcriptionfactors are described, for example, in U.S. Pat. Nos. 6,453,242;6,503,717; 6,534,261; 6,599,692; 6,607,882; 6,689,558; 6,824,978;6,933,113; 6,979,539; 7,013,219; and 7,163,824, the disclosures of allof which are incorporated by reference herein in their entireties.

TALE-protein fusions as described herein may also be delivered usingvectors containing sequences encoding one or more of the TALE-proteinfusions. Any vector systems may be used including, but not limited to,plasmid vectors, retroviral vectors, lentiviral vectors, adenovirusvectors, poxvirus vectors; herpesvirus vectors and adeno-associatedvirus vectors, etc. See, also, U.S. Pat. Nos. 6,534,261; 6,607,882;6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824, incorporatedby reference herein in their entireties. Furthermore, it will beapparent that any of these vectors may comprise one or more TALE-proteinfusions encoding sequences. Thus, when one or more TALE-protein fusions(e.g., a pair of TALENs) are introduced into the cell, the TALE-proteinfusions may be carried on the same vector or on different vectors. Whenmultiple vectors are used, each vector may comprise a sequence encodingone or multiple TALE-protein fusions.

Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids encoding engineered TALE-protein fusions incells (e.g. mammalian cells) whole organisms or target tissues. Suchmethods can also be used to administer nucleic acids encodingTALE-protein fusions to cells in vitro. In certain embodiments, nucleicacids encoding TALE protein fusions are administered for in vivo or exvivo uses. Non-viral vector delivery systems include DNA plasmids, nakednucleic acid, and nucleic acid complexed with a delivery vehicle such asa liposome or poloxamer. Viral vector delivery systems include DNA andRNA viruses, which have either episomal or integrated genomes afterdelivery to the cell. For a review of in vivo delivery of engineeredDNA-binding proteins and fusion proteins comprising these bindingproteins, see, e.g., Rebar (2004) Expert Opinion Invest. Drugs13(7):829-839; Rossi et al. (2007) Nature Biotech. 25(12):1444-1454 aswell as general gene delivery references such as Anderson, Science256:808-813 (1992); Nabel & Felgner, TIBTECH 11:211-217 (1993); Mitani &Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993);Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology6(10):1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin51(1):31-44 (1995); Haddada et al., in Current Topics in Microbiologyand Immunology Doerfler and Böhm (eds.) (1995); and Yu et al., GeneTherapy 1:13-26 (1994).

Non-viral vector delivery systems include electroporation, lipofection,microinjection, biolistics, virosomes, liposomes, immunoliposomes,polycation or lipid:nucleic acid conjugates, naked DNA, artificialvirions, and agent-enhanced uptake of DNA. Sonoporation using, e.g., theSonitron 2000 system (Rich-Mar) can also be used for delivery of nucleicacids. Viral vector delivery systems include DNA and RNA viruses, whichhave either episomal or integrated genomes after delivery to the cell.Additional exemplary nucleic acid delivery systems include thoseprovided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc.(Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.) andCopernicus Therapeutics Inc, (see for example U.S. Pat. No. 6,008,336).Lipofection is described in e.g., U.S. Pat. No. 5,049,386, U.S. Pat. No.4,946,787; and U.S. Pat. No. 4,897,355) and lipofection reagents aresold commercially (e.g., Transfectam™ and Lipofectin™). Cationic andneutral lipids that are suitable for efficient receptor-recognitionlipofection of polynucleotides include those of Felgner, WO 91/17424, WO91/16024. Delivery can be to cells (ex vivo administration) or targettissues (in vivo administration).

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

Additional methods of delivery include the use of packaging the nucleicacids to be delivered into EnGeneIC delivery vehicles (EDVs). These EDVsare specifically delivered to target tissues using bispecific antibodieswhere one arm of the antibody has specificity for the target tissue andthe other has specificity for the EDV. The antibody brings the EDVs tothe target cell surface and then the EDV is brought into the cell byendocytosis. Once in the cell, the contents are released (see MacDiarmidet al (2009) Nature Biotechnology vol 27(7) p. 643).

Suitable cells include but are not limited to eukaryotic and prokaryoticcells and/or cell lines. Non-limiting examples of such cells or celllines generated from such cells include COS, CHO (e.g., CHO-S, CHO-K1,CHO-DG44, CHO-DUXB11, CHO-DUKX, CHOK1SV), VERO, MDCK, WI38, V79,B14AF28-G3, BHK, HaK, NS0, SP2/0-Ag14, HeLa, HEK293 (e.g., HEK293-F,HEK293-H, HEK293-T), and perC6 cells as well as insect cells such asSpodoptera fugiperda (Sf), or fungal cells such as Saccharomyces, Pichiaand Schizosaccharomyces. In certain embodiments, the cell line is aCHO-K1, MDCK or HEK293 cell line. Additionally, primary cells may beisolated and used ex vivo for reintroduction into the subject to betreated following treatment with the TALE-fusions. Suitable primarycells include peripheral blood mononuclear cells (PBMC), and other bloodcell subsets such as, but not limited to, CD4+ T cells or CD8+ T cells.Suitable cells also include stem cells such as, by way of example,embryonic stem cells, induced pluripotent stem cells, hematopoietic stemcells, neuronal stem cells, mesenchymal stem cells, muscle stem cellsand skin stem cells.

Stem cells that have been modified may also be used in some embodiments.For example, stem cells that have been made resistant to apoptosis maybe used as therapeutic compositions where the stem cells also containthe TALE-fusion proteins of the invention. Resistance to apoptosis maycome about, for example, by knocking out BAX and/or BAK using BAX- orBAK-specific TALENs in the stem cells, or those that are disrupted in acaspase, again using caspase-6 specific TALENs for example.

Methods for introduction of DNA into hematopoietic stem cells aredisclosed, for example, in U.S. Pat. No. 5,928,638. Vectors useful forintroduction of transgenes into hematopoietic stem cells, e.g., CD34⁺cells, include adenovirus Type 35.

Vectors suitable for introduction of polynucleotides as described hereininclude described herein include non-integrating lentivirus vectors(IDLV). See, for example, Ory et al. (1996) Proc. Natl. Acad. Sci. USA93:11382-11388; Dull et al. (1998) J. Virol. 72:8463-8471; Zuffery etal. (1998) J. Virol. 72:9873-9880; Follenzi et al. (2000) NatureGenetics 25:217-222; U.S. Patent Publication No 2009/054985. As notedabove, the disclosed methods and compositions can be used in any type ofcell. Progeny, variants and derivatives of animal cells can also beused.

DNA constructs may be introduced into (e.g., into the genome of) adesired plant host by a variety of conventional techniques. For reviewsof such techniques see, for example, Weissbach & Weissbach Methods forPlant Molecular Biology (1988, Academic Press, N.Y.) Section VIII, pp.421-463; and Grierson & Corey, Plant Molecular Biology (1988, 2d Ed.),Blackie, London, Ch. 7-9.

For example, the DNA construct may be introduced directly into thegenomic DNA of the plant cell using techniques such as electroporationand microinjection of plant cell protoplasts, or the DNA constructs canbe introduced directly to plant tissue using biolistic methods, such asDNA particle bombardment (see, e.g., Klein et al (1987) Nature327:70-73). Alternatively, the DNA constructs may be combined withsuitable T-DNA flanking regions and introduced into a conventionalAgrobacterium tumefaciens host vector. Agrobacteriumtumefaciens-mediated transformation techniques, including disarming anduse of binary vectors, are well described in the scientific literature.See, for example Horsch et al (1984) Science 233:496-498, and Fraley etal (1983) Proc. Nat'l. Acad. Sci. USA 80:4803.

In addition, gene transfer may be achieved using non-Agrobacteriumbacteria or viruses such as Rhizobium sp. NGR234, Sinorhizoboiummeliloti, Mesorhizobium loti, potato virus X, cauliflower mosaic virusand cassava vein mosaic virus and/or tobacco mosaic virus, See, e.g.,Chung et al. (2006) Trends Plant Sci. 11(1):1-4.

The virulence functions of the Agrobacterium tumefaciens host willdirect the insertion of the construct and adjacent marker into the plantcell DNA when the cell is infected by the bacteria using binary T DNAvector (Bevan (1984) Nuc. Acid Res. 12:8711-8721) or the co-cultivationprocedure (Horsch et al (1985) Science 227:1229-1231). Generally, theAgrobacterium transformation system is used to engineer dicotyledonousplants (Bevan et al (1982) Ann. Rev. Genet 16:357-384; Rogers et al(1986) Methods Enzymol. 118:627-641). The Agrobacterium transformationsystem may also be used to transform, as well as transfer, DNA tomonocotyledonous plants and plant cells. See U.S. Pat. No. 5,591,616;Hernalsteen et al (1984) EMBO J 3:3039-3041; Hooykass-Van Slogteren etal (1984) Nature 311:763-764; Grimsley et al (1987) Nature 325:1677-179;Boulton et al (1989) Plant Mol. Biol. 12:31-40; and Gould et al (1991)Plant Physiol. 95:426-434.

Alternative gene transfer and transformation methods include, but arenot limited to, protoplast transformation through calcium-, polyethyleneglycol (PEG)- or electroporation-mediated uptake of naked DNA (seePaszkowski et al. (1984) EMBO J 3:2717-2722, Potrykus et al. (1985)Molec. Gen. Genet. 199:169-177; Fromm et al. (1985) Proc. Nat. Acad.Sci. USA 82:5824-5828; and Shimamoto (1989) Nature 338:274-276) andelectroporation of plant tissues (D'Halluin et al. (1992) Plant Cell4:1495-1505). Additional methods for plant cell transformation includemicroinjection, silicon carbide mediated DNA uptake (Kaeppler et al.(1990) Plant Cell Reporter 9:415-418), and microprojectile bombardment(see Klein et al. (1988) Proc. Nat. Acad. Sci. USA 85:4305-4309; andGordon-Kamm et al. (1990) Plant Cell 2:603-618).

Organisms

The methods and compositions described herein are applicable to anyorganism in which it is desired to regulate gene expression and/or alterthe organism through genomic modification, including but not limited toeukaryotic organisms such as plants, animals (e.g., mammals such asmice, rats, primates, farm animals, rabbits, etc.), fish, and the like.Eukaryotic (e.g., yeast, plant, fungal, piscine and mammalian cells suchas feline, canine, murine, bovine, ovine, and porcine) cells can beused. Cells from organisms containing one or more homozygous KO loci asdescribed herein or other genetic modifications can also be used.

Exemplary mammalian cells include any cell or cell line of the organismof interest, for example oocytes, K562 cells, CHO (Chinese hamsterovary) cells, HEP-G2 cells, BaF-3 cells, Schneider cells, COS cells(monkey kidney cells expressing SV40 T-antigen), CV-1 cells, HuTu80cells, NTERA2 cells, NB4 cells, HL-60 cells and HeLa cells, 293 cells(see, e.g., Graham et al. (1977) J. Gen. Virol. 36:59), and myelomacells like SP2 or NS0 (see, e.g., Galfre and Milstein (1981) Meth.Enzymol. 73(B):3 46). Peripheral blood mononucleocytes (PBMCs) orT-cells can also be used, as can embryonic and adult stem cells. Forexample, stem cells that can be used include embryonic stem cells (ES),induced pluripotent stem cells (iPSC), mesenchymal stem cells,hematopoietic stem cells, liver stem cells, skin stem cells and neuronalstem cells.

Exemplary target plants and plant cells include, but are not limited to,those monocotyledonous and dicotyledonous plants, such as cropsincluding grain crops (e.g., wheat, maize, rice, millet, barley), fruitcrops (e.g., tomato, apple, pear, strawberry, orange), forage crops(e.g., alfalfa), root vegetable crops (e.g., carrot, potato, sugarbeets, yam), leafy vegetable crops (e.g., lettuce, spinach); vegetativecrops for consumption (e.g. soybean and other legumes, squash, peppers,eggplant, celery etc), flowering plants (e.g., petunia, rose,chrysanthemum), conifers and pine trees (e.g., pine fir, spruce); poplartrees (e.g. P. tremula×P. alba); fiber crops (cotton, jute, flax,bamboo) plants used in phytoremediation (e.g., heavy metal accumulatingplants); oil crops (e.g., sunflower, rape seed) and plants used forexperimental purposes (e.g., Arabidopsis). Thus, the disclosed methodsand compositions have use over a broad range of plants, including, butnot limited to, species from the genera Asparagus, Avena, Brassica,Citrus, Citrullus, Capsicum, Cucurbita, Daucus, Erigeron, Glycine,Gossypium, Hordeum, Lactuca, Lolium, Lycopersicon, Malus, Manihot,Nicotiana, Orychophragmus, Oryza, Persea, Phaseolus, Pisum, Pyrus,Prunus, Raphanus, Secale, Solanum, Sorghum, Triticum, Vitis, Vigna, andZea. The term plant cells include isolated plant cells as well as wholeplants or portions of whole plants such as seeds, callus, leaves, roots,etc. The present disclosure also encompasses seeds of the plantsdescribed above wherein the seed has the transgene or gene constructand/or has been modified using the compositions and/or methods describedherein. The present disclosure further encompasses the progeny, clones,cell lines or cells of the transgenic plants described above whereinsaid progeny, clone, cell line or cell has the transgene or geneconstruct.

Algae are being increasingly utilized for manufacturing compounds ofinterest, i.e. biofuels, plastics, hydrocarbons etc. Exemplary algaespecies include microalgae including diatoms and cyanobacteria as wellas Botryococcus braunii, Chlorella, Dunaliella tertiolecta, Gracileria,Pleurochrysis carterae, Sorgassum and Ulva.

Assays for Determining Regulation of Gene Expression by TALE fusionproteins

A variety of assays can be used to determine the level of geneexpression regulation by TALE-fusion proteins. The activity of aparticular TALE-fusion proteins can be assessed using a variety of invitro and in vivo assays, by measuring, e.g., protein or mRNA levels,product levels, enzyme activity, tumor growth; transcriptionalactivation or repression of a reporter gene; second messenger levels(e.g., cGMP, cAMP, IP3, DAG, Ca.sup.2+); cytokine and hormone productionlevels; and neovascularization, using, e.g., immunoassays (e.g., ELISAand immunohistochemical assays with antibodies), hybridization assays(e.g., RNase protection, northems, in situ hybridization,oligonucleotide array studies), colorimetric assays, amplificationassays, enzyme activity assays, tumor growth assays, phenotypic assays,and the like.

TALE-fusion proteins are typically first tested for activity in vitrousing cultured cells, e.g., 293 cells, CHO cells, VERO cells, BHK cells,HeLa cells, COS cells, plant cell lines, plant callous cultures and thelike. Preferably, human cells are used. The TALE-fusion protein is oftenfirst tested using a transient expression system with a reporter gene,and then regulation of the target endogenous gene is tested in cells andin animals, both in vivo and ex vivo. The TALE fusion proteins can berecombinantly expressed in a cell, recombinantly expressed in cellstransplanted into an animal or plant, or recombinantly expressed in atransgenic animal or plant, as well as administered as a protein to ananimal, plant or cell using delivery vehicles described herein. Thecells can be immobilized, be in solution, be injected into an animal, orbe naturally occurring in a transgenic or non-transgenic animal.

Modulation of gene expression is tested using one of the in vitro or invivo assays described herein. Samples or assays are treated with aTALE-fusion proteins and compared to control samples without the testcompound, to examine the extent of modulation.

The effects of the TALE-fusion proteins can be measured by examining anyof the parameters described above. Any suitable gene expression,phenotypic, or physiological change can be used to assess the influenceof a TALE-fusion protein. When the functional consequences aredetermined using intact cells or animals, one can also measure a varietyof effects such as tumor growth, neovascularization, hormone release,transcriptional changes to both known and uncharacterized geneticmarkers (e.g., northern blots or oligonucleotide array studies), changesin cell metabolism such as cell growth or pH changes, and changes inintracellular second messengers such as cGMP.

Preferred assays for TALE-fusion protein mediated regulation ofendogenous gene expression can be performed in vitro. In one preferredin vitro assay format, TALE-fusion protein mediated regulation ofendogenous gene expression in cultured cells is measured by examiningprotein production using an ELISA assay. The test sample is compared tocontrol cells treated with an empty vector or an unrelated TALE-fusionprotein that is targeted to another gene.

In another embodiment, TALE-fusion protein-mediated regulation ofendogenous gene expression is determined in vitro by measuring the levelof target gene mRNA expression. The level of gene expression is measuredusing amplification, e.g., using PCR, LCR, or hybridization assays,e.g., northern hybridization, RNase protection, dot blotting. RNaseprotection is used in one embodiment. The level of protein or mRNA isdetected using directly or indirectly labeled detection agents, e.g.,fluorescently or radioactively labeled nucleic acids, radioactively orenzymatically labeled antibodies, and the like, as described herein.

Alternatively, a reporter gene system can be devised using the targetgene promoter operably linked to a reporter gene such as luciferase,green fluorescent protein, CAT, or beta-gal. The reporter construct istypically co-transfected into a cultured cell. After treatment with theTALE-fusion proteins of choice, the amount of reporter genetranscription, translation, or activity is measured according tostandard techniques known to those of skill in the art.

Another example of a preferred assay format useful for monitoringTALE-fusion protein mediated regulation of endogenous gene expression isperformed in vivo. This assay is particularly useful for examiningTALE-fusions that inhibit expression of tumor promoting genes, genesinvolved in tumor support, such as neovascularization (e.g., VEGF), orthat activate tumor suppressor genes such as p53. In this assay,cultured tumor cells expressing the TALE-fusions of choice are injectedsubcutaneously into an immune compromised mouse such as an athymicmouse, an irradiated mouse, or a SCID mouse. After a suitable length oftime, preferably 4-8 weeks, tumor growth is measured, e.g., by volume orby its two largest dimensions, and compared to the control. Tumors thathave statistically significant reduction (using, e.g., Student's T test)are said to have inhibited growth. Alternatively, the extent of tumorneovascularization can also be measured. Immunoassays using endothelialcell specific antibodies are used to stain for vascularization of thetumor and the number of vessels in the tumor. Tumors that have astatistically significant reduction in the number of vessels (using,e.g., Student's T test) are said to have inhibited neovascularization.

Transgenic and non-transgenic plants or animals as described above arealso used as a preferred embodiment for examining regulation ofendogenous gene expression in vivo. Transgenic organisms typicallyexpress the TALE-fusions of choice. Alternatively, organisms thattransiently express the TALE-fusions of choice, or to which the TALEfusion proteins have been administered in a delivery vehicle, can beused. Regulation of endogenous gene expression is tested using any oneof the assays described herein.

Nucleic Acids Encoding TALE-fusion proteins

Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids encoding engineered TALE domain fusions inmammalian cells, in whole organisms or in target tissues. Such methodscan be used to administer nucleic acids encoding TALE domain fusions tocells in vitro. Preferably, the nucleic acids encoding TALE domainfusions are administered for in vivo or ex vivo uses. Non-viral vectordelivery systems include DNA plasmids, naked nucleic acid, and nucleicacid complexed with a delivery vehicle such as a liposome. Viral vectordelivery systems include DNA and RNA viruses, which have either episomalor integrated genomes after delivery to the cell. For a review of genetherapy procedures, see Anderson, Science 256:808-813 (1992); Nabel &Felgner, TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166(1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460(1992); Van Brunt, Biotechnology 6(10):1149-1154 (1988); Vigne,Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer &Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada etal., in Current Topics in Microbiology and Immunology Doerfler and Bohm(eds) (1995); and Yu et al., Gene Therapy 1:13-26 (1994).

The use of RNA or DNA viral based systems for the delivery of nucleicacids encoding engineered TALE domain fusions takes advantage of highlyevolved processes for targeting a virus to specific cells in the bodyand trafficking the viral payload to the nucleus. Viral vectors can beadministered directly to patients (in vivo) or they can be used to treatcells in vitro and the modified cells are administered to patients (exvivo). Conventional viral based systems for the delivery of TALE domainfusions could include retroviral, lentivirus, adenoviral,adeno-associated and herpes simplex virus vectors, for gene transfer.Viral vectors are currently the most efficient and versatile method ofgene transfer in target cells and tissues. Integration in the hostgenome is possible with the retrovirus, lentivirus, and adeno-associatedvirus gene transfer methods, often resulting in long term expression ofthe inserted transgene. Additionally, high transduction efficiencieshave been observed in many different cell types and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vector that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system would thereforedepend on the target tissue. Retroviral vectors are comprised ofcis-acting long terminal repeats with packaging capacity for up to 6-10kb of foreign sequence. The minimum cis-acting LTRs are sufficient forreplication and packaging of the vectors, which are then used tointegrate the therapeutic gene into the target cell to provide permanenttransgene expression. Widely used retroviral vectors include those basedupon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV),Simian Immuno deficiency virus (SIV), human immuno deficiency virus(HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommerfelt et al, Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700).

In applications where transient expression of the TALE domain fusions ispreferred, adenoviral based systems are typically used. Adenoviral basedvectors are capable of very high transduction efficiency in many celltypes and do not require cell division. With such vectors, high titerand levels of expression have been obtained. This vector can be producedin large quantities in a relatively simple system. Adeno-associatedvirus (“AAV”) vectors are also used to transduce cells with targetnucleic acids, e.g., in the in vitro production of nucleic acids andpeptides, and for in vivo and ex vivo gene therapy procedures (see,e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368;WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J.Clin. Invest. 94:1351 (1994). Construction of recombinant AAV vectorsare described in a number of publications, including U.S. Pat. No.5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985);Tratschin, et al., Mol Cell. Biol. 4:2072-2081 (1984); Hermonat &Muzyczka, Proc Natl Acad Sci USA 81:6466-6470 (1984); and Samulski etal., J. Virol. 63:03822-3828 (1989).

In particular, at least six viral vector approaches are currentlyavailable for gene transfer in clinical trials, with retroviral vectorsby far the most frequently used system. All of these viral vectorsutilize approaches that involve complementation of defective vectors bygenes inserted into helper cell lines to generate the transducing agent.

pLASN and MFG-S are examples are retroviral vectors that have been usedin clinical trials (Dunbar et al., Blood 85:3048-305 (1995); Kohn etal., Nat. Med. 1:1017-102 (1995); Malech et al., Proc Natl Acad Sci USA94:22 12133-12138 (1997)). PA317/pLASN was the first therapeutic vectorused in a gene therapy trial. (Blaese et al., Science 270:475480(1995)). Transduction efficiencies of 50% or greater have been observedfor MFG-S packaged vectors. (Ellem et al., Immunol Immunother.44(1):10-20 (1997); Dranoff et al., Hum. Gene Ther. 1:111-2 (1997).

Recombinant adeno-associated virus vectors (rAAV) are a promisingalternative to gene delivery systems based on the defective andnonpathogenic parvovirus adeno-associated type 2 virus. All vectors arederived from a plasmid that retains only the AAV 145 bp invertedterminal repeats flanking the transgene expression cassette. Efficientgene transfer and stable transgene delivery due to integration into thegenomes of the transduced cell are key features for this vector system.(Wagner et al., Lancet 351:9117 1702-3 (1998), Kearns et al., Gene Ther.9:748-55 (1996)).

Replication-deficient recombinant adenoviral vectors (Ad) arepredominantly used for colon cancer gene therapy, because they can beproduced at high titer and they readily infect a number of differentcell types. Most adenovirus vectors are engineered such that a transgenereplaces the Ad E1a, E1b, and E3 genes; subsequently the replicationdefector vector is propagated in human 293 cells that supply deletedgene function in trans. Ad vectors can transduce multiply types oftissues in vivo, including nondividing, differentiated cells such asthose found in the liver, kidney and muscle system tissues. ConventionalAd vectors have a large carrying capacity. An example of the use of anAd vector in a clinical trial involved polynucleotide therapy forantitumor immunization with intramuscular injection (Sterman et al.,Hum. Gene Ther. 7:1083-9 (1998)). Additional examples of the use ofadenovirus vectors for gene transfer include Rosenecker et al, Infection24:1 5-10 (1996); Sterman et al., Hum. Gene Ther. 9:7 1083-1089 (1998);Welsh et al., Hum. Gene Ther. 2:205-18 (1995); Alvarez et al., Hum. GeneTher. 5:597-613 (1997); Topf et al., Gene Ther. 5:507-513 (1998);Sterman et al., Hum. Gene Ther. 7:1083-1089 (1998); U.S. PatentPublication No. 2008/0159996.

Packaging cells are used to form virus particles that are capable ofinfecting a host cell. Such cells include 293 cells, which packageadenovirus, and psi2 cells or PA317 cells, which package retrovirus.Viral vectors used in gene therapy are usually generated by producercell line that packages a nucleic acid vector into a viral particle. Thevectors typically contain the minimal viral sequences required forpackaging and subsequent integration into a host, other viral sequencesbeing replaced by an expression cassette for the protein to beexpressed. The missing viral functions are supplied in trans by thepackaging cell line. For example, AAV vectors used in gene therapytypically only possess ITR sequences from the AAV genome, which arerequired for packaging and integration into the host genome. Viral DNAis packaged in a cell line, which contains a helper plasmid encoding theother AAV genes, namely rep and cap, but lacking ITR sequences. The cellline is also infected with adenovirus as a helper. The helper viruspromotes replication of the AAV vector and expression of AAV genes fromthe helper plasmid. The helper plasmid is not packaged in significantamounts due to a lack of ITR sequences. Contamination with adenoviruscan be reduced by, e.g., heat treatment to which adenovirus is moresensitive than AAV.

In many gene therapy applications, it is desirable that the gene therapyvector be delivered with a high degree of specificity to a particulartissue type. A viral vector is typically modified to have specificityfor a given cell type by expressing a ligand as a fusion protein with aviral coat protein on the viruses outer surface. The ligand is chosen tohave affinity for a receptor known to be present on the cell type ofinterest. For example, Han et al., Proc Natl Acad Sci USA 92:9747-9751(1995), reported that Moloney murine leukemia virus can be modified toexpress human heregulin fused to gp70, and the recombinant virus infectscertain human breast cancer cells expressing human epidermal growthfactor receptor. This principle can be extended to other pairs of virus,expressing a ligand fusion protein and target cell expressing areceptor. For example, filamentous phage can be engineered to displayantibody fragments (e.g., FAB or Fv) having specific binding affinityfor virtually any chosen cellular receptor. Although the abovedescription applies primarily to viral vectors, the same principles canbe applied to nonviral vectors. Such vectors can be engineered tocontain specific uptake sequences thought to favor uptake by specifictarget cells.

Gene therapy vectors can be delivered in vivo by administration to anindividual patient, typically by systemic administration (e.g.,intravenous, intraperitoneal, intramuscular, subdermal, or intracranialinfusion) or topical application, as described below. Alternatively,vectors can be delivered to cells ex vivo, such as cells explanted froman individual patient (e.g., lymphocytes, bone marrow aspirates, tissuebiopsy) or universal donor hematopoietic stem cells, followed byreimplantation of the cells into a patient, usually after selection forcells which have incorporated the vector.

Ex vivo cell transfection for diagnostics, research, or for gene therapy(e.g., via re-infusion of the transfected cells into the host organism)is well known to those of skill in the art. In a preferred embodiment,cells are isolated from the subject organism, transfected with a TALEfusion nucleic acid (gene or cDNA), and re-infused back into the subjectorganism (e.g., patient). Various cell types suitable for ex vivotransfection are well known to those of skill in the art (see, e.g.,Freshney et al., Culture of Animal Cells, A Manual of Basic Technique(3rd ed. 1994)) and the references cited therein for a discussion of howto isolate and culture cells from patients).

In one embodiment, stem cells are used in ex vivo procedures for celltransfection and gene therapy. The advantage to using stem cells is thatthey can be differentiated into other cell types in vitro, or can beintroduced into a mammal (such as the donor of the cells) where theywill engraft in the bone marrow. Methods for differentiating CD34+ cellsin vitro into clinically important immune cell types using cytokinessuch a GM-CSF, IFN-.gamma. and TNF-alpha are known (see Inaba et al., J.Exp. Med. 176:1693-1702 (1992)).

Stem cells are isolated for transduction and differentiation using knownmethods. For example, stem cells are isolated from bone marrow cells bypanning the bone marrow cells with antibodies which bind unwanted cells,such as CD4+ and CD8+ (T cells), CD45+ (panb cells), GR-1(granulocytes), and Iad (differentiated antigen presenting cells) (seeInaba et al., J. Exp. Med. 176:1693-1702 (1992)). Exemplary stem cellsinclude human embryonic stem cells (hES), induced pluripotent stem cells(iPSC), hematopoietic stem cells, mesenchymal stem cells, neuronal stemcells, and muscle stem cells.

Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containingtherapeutic TALE domain fusion nucleic acids can be also administereddirectly to the organism for transduction of cells in vivo.Alternatively, naked DNA can be administered. Administration is by anyof the routes normally used for introducing a molecule into ultimatecontact with blood or tissue cells. Suitable methods of administeringsuch nucleic acids are available and well known to those of skill in theart, and, although more than one route can be used to administer aparticular composition, a particular route can often provide a moreimmediate and more effective reaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention, as described below (see, e.g., Remington'sPharmaceutical Sciences, 17th ed., 1989).

Pharmaceutical Compositions and Administration

TALE-fusions and expression vectors encoding TALE fusions can beadministered directly to the patient for modulation of gene expressionand for therapeutic or prophylactic applications, for example, cancer,ischemia, diabetic retinopathy, macular degeneration, rheumatoidarthritis, psoriasis, HIV infection, sickle cell anemia, Alzheimer'sdisease, muscular dystrophy, neurodegenerative diseases, vasculardisease, cystic fibrosis, stroke, and the like. Examples ofmicroorganisms that can be inhibited by TALE fusion protein gene therapyinclude pathogenic bacteria, e.g., chlamydia, rickettsial bacteria,mycobacteria, staphylococci, streptococci, pneumococci, meningococci andconococci, klebsiella, proteus, serratia, pseudomonas, legionella,diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax,plague, leptospirosis, and Lyme disease bacteria; infectious fungus,e.g., Aspergillus, Candida species; protozoa such as sporozoa (e.g.,Plasmodia), rhizopods (e.g., Entamoeba) and flagellates (Trypanosoma,Leishmania, Trichomonas, Giardia, etc.); viral diseases, e.g., hepatitis(A, B, or C), herpes virus (e.g. VZV, HSV-1, HSV-6, HSV-II, CMV, andEBV), HIV, Ebola, adenovirus, influenza virus, flaviviruses, echovirus,rhinovirus, coxsackie virus, comovirus, respiratory syncytial virus,mumps virus, rotavirus, measles virus, rubella virus, parvovirus,vaccinia virus, HTLV virus, dengue virus, papillomavirus, poliovirus,rabies virus, and arboviral encephalitis virus, etc.

Administration of therapeutically effective amounts is by any of theroutes normally used for introducing TALE-fusions into ultimate contactwith the tissue to be treated. The TALE-fusions are administered in anysuitable manner, preferably with pharmaceutically acceptable carriers.Suitable methods of administering such modulators are available and wellknown to those of skill in the art, and, although more than one routecan be used to administer a particular composition, a particular routecan often provide a more immediate and more effective reaction thananother route.

Formulations suitable for parenteral administration, such as, forexample, by intravenous, intramuscular, intradermal, and subcutaneousroutes, include aqueous and non-aqueous, isotonic sterile injectionsolutions, which can contain antioxidants, buffers, bacteriostats, andsolutes that render the formulation isotonic with the blood of theintended recipient, and aqueous and non-aqueous sterile suspensions thatcan include suspending agents, solubilizers, thickening agents,stabilizers, and preservatives. In the practice of this invention,compositions can be administered, for example, by intravenous infusion,orally, topically, intraperitoneally, intravesically or intrathecally.The formulations of compounds can be presented in unit-dose ormulti-dose sealed containers, such as ampules and vials. Injectionsolutions and suspensions can be prepared from sterile powders,granules, and tablets of the kind previously described.

Regulation of Gene Expression in Plants

TALE-fusions can be used to engineer plants for traits such as increaseddisease resistance, modification of structural and storagepolysaccharides, flavors, proteins, and fatty acids, fruit ripening,yield, color, nutritional characteristics, improved storage capability,drought or submergence/flood tolerance, and the like. In particular, theengineering of crop species for enhanced oil production, e.g., themodification of the fatty acids produced in oilseeds, is of interest.See, e.g., U.S. Pat. No. 7,262,054; and U.S. Patent Publication Nos.2008/0182332 and 20090205083:

Seed oils are composed primarily of triacylglycerols (TAGs), which areglycerol esters of fatty acids. Commercial production of these vegetableoils is accounted for primarily by six major oil crops (soybean, oilpalm, rapeseed, sunflower, cotton seed, and peanut.) Vegetable oils areused predominantly (90%) for human consumption as margarine, shortening,salad oils, and flying oil. The remaining 10% is used for non-foodapplications such as lubricants, oleochemicals, biofuels, detergents,and other industrial applications.

The desired characteristics of the oil used in each of theseapplications varies widely, particularly in terms of the chain lengthand number of double bonds present in the fatty acids making up theTAGs. These properties are manipulated by the plant in order to controlmembrane fluidity and temperature sensitivity. The same properties canbe controlled using TALE domain fusions to produce oils with improvedcharacteristics for food and industrial uses.

The primary fatty acids in the TAGs of oilseed crops are 16 to 18carbons in length and contain 0 to 3 double bonds. Palmitic acid (16:0[16 carbons: 0 double bonds]), oleic acid (18:1), linoleic acid (18:2),and linolenic acid (18:3) predominate. The number of double bonds, ordegree of saturation, determines the melting temperature, reactivity,cooking performance, and health attributes of the resulting oil.

The enzyme responsible for the conversion of oleic acid (18:1) intolinoleic acid (18:2) (which is then the precursor for 18:3 formation) isDELTA12-oleate desaturase, also referred to as omega-6 desaturase. Ablock at this step in the fatty acid desaturation pathway should resultin the accumulation of oleic acid at the expense of polyunsaturates.

In one embodiment proteins containing TALE domain(s) are used toregulate expression of the FAD2-1 gene in soybeans. Two genes encodingmicrosomal DELTA.6 desaturases have been cloned recently from soybean,and are referred to as FAD2-1 and FAD2-2 (Heppard et al., Plant Physiol.110:311-319 (1996)). FAD2-1 (delta 12 desaturase) appears to control thebulk of oleic acid desaturation in the soybean seed. TALE-fusions canthus be used to modulate gene expression of FAD2-1 in plants.Specifically, TALE domain fusions can be used to inhibit expression ofthe FAD2-1 gene in soybean in order to increase the accumulation ofoleic acid (18:1) in the oil seed. Moreover, TALE-fusions can be used tomodulate expression of any other plant gene, such as delta-9 desaturase,delta-12 desaturases from other plants, delta-15 desaturase, acetyl-CoAcarboxylase, acyl-ACP-thioesterase, ADP-glucose pyrophosphorylase,starch synthase, cellulose synthase, sucrose synthase,senescence-associated genes, heavy metal chelators, fatty acidhydroperoxide lyase, polygalacturonase, EPSP synthase, plant viralgenes, plant fungal pathogen genes, and plant bacterial pathogen genes.

Functional Genomics Assays

TALE-fusions also have use for assays to determine the phenotypicconsequences and function of gene expression. The recent advances inanalytical techniques, coupled with focussed mass sequencing effortshave created the opportunity to identify and characterize many moremolecular targets than were previously available. This new informationabout genes and their functions will speed along basic biologicalunderstanding and present many new targets for therapeutic intervention.In some cases analytical tools have not kept pace with the generation ofnew data. An example is provided by recent advances in the measurementof global differential gene expression. These methods, typified by geneexpression microarrays, differential cDNA cloning frequencies,subtractive hybridization and differential display methods, can veryrapidly identify genes that are up or down-regulated in differenttissues or in response to specific stimuli. Increasingly, such methodsare being used to explore biological processes such as, transformation,tumor progression, the inflammatory response, neurological disordersetc. One can now very easily generate long lists of differentiallyexpressed genes that correlate with a given physiological phenomenon,but demonstrating a causative relationship between an individualdifferentially expressed gene and the phenomenon is difficult. Untilnow, simple methods for assigning function to differentially expressedgenes have not kept pace with the ability to monitor differential geneexpression.

Using conventional molecular approaches, over expression of a candidategene can be accomplished by cloning a full-length cDNA, subcloning itinto a mammalian expression vector and transfecting the recombinantvector into an appropriate host cell. This approach is straightforwardbut labor intensive, particularly when the initial candidate gene isrepresented by a simple expressed sequence tag (EST). Under expressionof a candidate gene by “conventional” methods is yet more problematic.Antisense methods and methods that rely on targeted ribozymes areunreliable, succeeding for only a small fraction of the targetsselected. Gene knockout by homologous recombination works fairly well inrecombinogenic stem cells but very inefficiently in somatically derivedcell lines. In either case large clones of syngeneic genomic DNA (on theorder of 10 kb) should be isolated for recombination to workefficiently.

The TALE-fusion technology can be used to rapidly analyze differentialgene expression studies. Engineered TALE domain fusions can be readilyused to up or down-regulate any endogenous target gene. Very littlesequence information is required to create a gene-specific DNA bindingdomain. This makes the TALE domain fusions technology ideal for analysisof long lists of poorly characterized differentially expressed genes.One can simply build a TALE-based DNA-binding domain for each candidategene, create chimeric up and down-regulating artificial transcriptionfactors and test the consequence of up or down-regulation on thephenotype under study (transformation, response to a cytokine etc.) byswitching the candidate genes on or off one at a time in a model system.

This specific example of using engineered TALE domain fusions to addfunctional information to genomic data is merely illustrative. Anyexperimental situation that could benefit from the specific up ordown-regulation of a gene or genes could benefit from the reliabilityand ease of use of engineered TALE-fusions.

Additionally, greater experimental control can be imparted by TALEdomain fusions than can be achieved by more conventional methods. Thisis because the production and/or function of an engineered TALE-fusionscan be placed under small molecule control. Examples of this approachare provided by the Tet-On system, the ecdysone-regulated system and asystem incorporating a chimeric factor including a mutant progesteronereceptor. These systems are all capable of indirectly imparting smallmolecule control on any endogenous gene of interest or any transgene byplacing the function and/or expression of a ZFP regulator under smallmolecule control.

Transgenic Organisms

A further application of the TALE-fusion technology is manipulating geneexpression and/or altering the genome to produce transgenic animals orplants. As with cell lines, over-expression of an endogenous gene or theintroduction of a heterologous gene to a transgenic animal, such as atransgenic mouse, is a fairly straightforward process. Similarly,production of transgenic plants is well known. The TALE domain fusionstechnology described herein can be used to readily generate transgenicanimals and plants.

The use of engineered TALE domain fusions to manipulate gene expressioncan be restricted to adult animals using the small molecule regulatedsystems described in the previous section. Expression and/or function ofa TALE domain-based repressor can be switched off during development andswitched on of will in the adult animals. This approach relies on theaddition of the TALE-fusions expressing module only; homologousrecombination is not required. Because the TALE domain fusionsrepressors are trans dominant, there is no concern about germlinetransmission or homozygosity. These issues dramatically affect the timeand labor required to go from a poorly characterized gene candidate (acDNA or EST clone) to a mouse model. This ability can be used to rapidlyidentify and/or validate gene targets for therapeutic intervention,generate novel model systems and permit the analysis of complexphysiological phenomena (development, hematopoiesis, transformation,neural function etc.) Chimeric targeted mice can be derived according toHogan et al., Manipulating the Mouse Embryo: A Laboratory Manual,(1988); Teratocarcinomas and Embryonic Stem Cells: A Practical Approach,Robertson, ed., (1987); and Capecchi et al., Science 244:1288 (1989).

Genetically modified animals may be generated by deliver of the nucleicacid encoding the TALE fusion into a cell or an embryo. Typically, theembryo is a fertilized one cell stage embryo. Delivery of the nucleicacid may be by any of the methods known in the art including microinjection into the nucleus or cytoplasm of the embryo. TALE fusionencoding nucleic acids may be co-delivered with donor nucleic acids asdesired. The embryos are then cultured as in known in the art to developa genetically modified animal.

In one aspect of the invention, genetically modified animals in which atleast one chromosomal sequence encoding a gene or locus of interest hasbeen edited are provided. For example, the edited gene may becomeinactivated such that it is not transcribed or properly translated.Alternatively, the sequence may be edited such that an alternate form ofthe gene is expressed (e.g. insertion (knock in) or deletion (knock out)of one or more amino acids in the expressed protein). In addition, thegene of interest may comprise an inserted sequence such as a regulatoryregion. The genetically modified animal may be homozygous for the editedsequence or may be heterozygous. In some embodiments, the geneticallymodified animal may have sequence inserted (knocked in) in a ‘safeharbor’ locus such as the Rosa26, HPRT, CCR5 or AAVS1 (PPP1R12C) loci.These knock in animals may be additionally edited at other chromosomalloci. In some embodiments, the sequences of interest are inserted intothe safe harbor without any selection markers, and/or without a promoterand so rely on the endogenous promoter to drive expression. In someaspects, the genetically modified animal may be “humanized” such thatcertain genes specific to the host species animal are replaced with thehuman homolog. In this way, genetically modified animals are producedwith a human gene expressed (e.g. Factor IX) to allow for thedevelopment of an animal model system to study the human gene, proteinor disease. In some embodiments, the gene of interest may furthercomprise a recombinase recognition site such as loxP or FRT forrecognition of the cognate recombinase Cre and FLP, respectively, whichcan flank the inserted gene(s) of interest. Genes may be insertedcontaining the nuclease sites such that crossing the geneticallymodified animal with another genetically modified animal expressing thecognate recombinase (e.g Cre) will result in progeny that lack theinserted gene.

Applications

The disclosed methods and compositions can be used to control generegulation at a desired locus. Genes of choice may be activated orrepressed, depending on the transcriptional regulatory domain that isfused to the TALE-repeat domain. TALE activators may be targeted topluripotency-inducing genes for the goal of producing iPSCs fromdifferentiated cells. This may be of use for in vitro and in vivo modeldevelopment for specific disease states and for developing celltherapeutics derived from iPSCs.

The TALE-fusions may be useful themselves as therapeutic agents,especially in immune privileged tissues such as in the brain or eye.Designed activators, for example, are especially useful for increasingthe dose of a gene product that requires natural splice variant ratiosfor, proper function (e.g. VEGF), or for genes that are toxic whereoverexpressed. Transient exposure to designed TALE regulators may alsoallow permanent switching of gene expression status via the use offunctional domain that impose epigenetic changes. This technology couldprovide additional utility for generating stem cells and controllingtheir differentiation pathways. Additionally, TALE-fusions may be of usein immunosuppressed patients.

The disclosed methods and compositions can also be used for genomicediting of any gene or genes. In certain applications, the methods andcompositions can be used for inactivation of genomic sequences. To date,cleavage-based methods have been used to target modifications to thegenomes of at least nine higher eukaryotes for which such capabilitieswere previously unavailable, including economically important speciessuch as corn and rat. In other applications, the methods andcompositions allow for generation of random mutations, includinggeneration of novel allelic forms of genes with different expression orbiological properties as compared to unedited genes or integration ofhumanized genes, which in turn allows for the generation of cell oranimal models. In other applications, the methods and compositions canbe used for creating random mutations at defined positions of genes thatallows for the identification or selection of animals carrying novelallelic forms of those genes. In other applications, the methods andcompositions allow for targeted integration of an exogenous (donor)sequence into any selected area of the genome. Regulatory sequences(e.g. promoters) could be integrated in a targeted fashion at a site ofinterest. By “integration” is meant both physical insertion (e.g., intothe genome of a host cell) and, in addition, integration by copying ofthe donor sequence into the host cell genome via the specialized nucleicacid information exchange process that occurs during homology-directedDNA repair.

Donor sequences can also comprise nucleic acids such as shRNAs, miRNAsetc. These small nucleic acid donors can be used to study their effectson genes of interest within the genome. Genomic editing (e.g.,inactivation, integration and/or targeted or random mutation) of ananimal gene can be achieved, for example, by a single cleavage event, bycleavage followed by non-homologous end joining, by cleavage followed byhomology-directed repair mechanisms, by cleavage followed by physicalintegration of a donor sequence, by cleavage at two sites followed byjoining so as to delete the sequence between the two cleavage sites, bytargeted recombination of a missense or nonsense codon into the codingregion, by targeted recombination of an irrelevant sequence (i.e., a“stuffer” sequence) into the gene or its regulatory region, so as todisrupt the gene or regulatory region, or by targeting recombination ofa splice acceptor sequence into an intron to cause mis-splicing of thetranscript. In some applications, transgenes of interest may beintegrated into a safe harbor locus within a mammalian or plant genomeusing TALEN-induced DSB at a specified location. See, U.S. PatentPublication Nos. 20030232410; 20050208489; 20050026157; 20050064474;20060188987; 20060063231; and International Publication WO 07/014,275,the disclosures of which are incorporated by reference in theirentireties for all purposes. These TALENs may also be supplied ascomponents of kits for targeted genetic manipulation.

TALE-repeat domains, optionally with novel or atypical RVDs, andmoreover optionally attached to N-cap and/or C-cap residues, can also befused to DNA manipulating enzymes such as recombinases, transposases,resolvases or integrases. Thus these domains can be used to maketargeted fusion proteins that would allow the development of such toolsand/or therapeutics as targeted transposons and the like. Additionally,a TALE-repeat domain, optionally attached to N-cap and C-cap residues,may be fused to nuclease domains to create designer restriction enzymes.For example, a TALE-repeat domain, optionally attached to N-cap andC-cap residues, may be fused to a single-chain FokI domain (wherein twoFokI cleavage half domains are joined together using a linker of choice)such that treatment of a DNA preparation with the nuclease fusion canallow cleavage to occur exactly at the desired location. This technologywould be useful for cloning and manipulation of DNA sequences that arenot readily approached with standard restriction enzymes. Such a systemwould also be useful in specialized cell systems used in manufacturing.For example, the CHO-derived cell lines do not have an endogenouslyactive transposase/integrase system. TALE-transposase/integrase systemscould be developed for specific targeting in CHO cells and could beuseful for knock out/knock in, genome editing etc due to the highlyspecific nature of the TALE DNA binding domain.

TALE-fusion proteins can be used to prevent binding of specificDNA-binding proteins to a given locus. For example, a natural regulatoryprotein may be blocked from binding to its natural target in a promotersimply because an engineered TALE protein has been expressed in the hostcell and it occupies the site on the DNA, thus preventing regulation bythe regulatory protein.

TALE-fusion proteins may be engineered to bind to RNA. In this way, forexample, splice donors and/or splice acceptor sites could be masked andwould prevent splicing at specific locations in a mRNA. In otheraspects, a TALE may be engineered to bind specific functional RNAs suchas shRNAs, miRNA or RNAis, for example.

TALE fusion proteins can be useful in diagnostics. For example, theproteins may be engineered to recognize certain sequences in the genometo identify alleles known to be associated with a specific disease. Forexample, TALE-fusions with a specified number of TALE repeat units maybe utilized as a “yard stick” of sorts to measure the number oftrinucleotide repeats in patients with the potential of having atrinucleotide repeat disorder (e.g. Huntingdon's Disease) to determinethe likelihood of becoming afflicted with one of these diseases or toprognosticate the severity of the symptoms. These fusion proteins mayalso be supplied as components of diagnostic kits to allow rapididentification of genomic markers of interest. Additionally, theseproteins may be purified from cells and used in diagnostic kits or fordiagnostic reagents for uses such as analyzing the allele type of a geneof interest, measuring mRNA expression levels etc. The TALE fusions maybe attached to silicon chips or beads for multichannel or microfluidicanalyses.

TALE fusions may be useful in manufacturing settings. TALE-transcriptionfactor fusions or TALENs may be used in cell lines of interest (e.g. CHOcells) or in algae (e.g. for biofuel production).

There are a variety of applications for TALE fusion proteins mediatedgenomic editing of a gene or genomic loci. The methods and compositionsdescribed herein allow for the generation of models of human diseasesand for plant crops with desired characteristics.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

EXAMPLES Example 1 Cloning of a Natural TALE from Xanthomonas axonopodis

To identify a natural TALE protein that could serve as an initial designframework, a canonical, natural TALE that both exhibited a high degreeof specificity as well as evidence of target sequence binding inmammalian cells was identified. Specifically, a TALE protein containing12.5 TALE repeats (12 full repeats and a half repeat referred to as TALE13) was cloned from Xanthomonas axonopodis by PCR amplification usingthe following primer pair: pthA_d152N_EcoR,ACGTGGATTCATGGTGGATCTACGCACGCTC (SEQ ID NO:52) and pthA_Sac2_Rev,TACGTCCGCGGTCCTGAGGCAATAGCTCCATCA (SEQ ID NO:53) The primer pair wasoriginally designed to amplify the AvrBs3 gene with the N-terminal 152amino acids truncation. It has been previously shown that thesesequences are necessary for transport into plant cells, but otherwiseare dispensable for function (see Szurek et al (2002) Mol. Micro 46(1)p. 13-23). Several TALE proteins, characterized by the highly conservedsequences with the variation of the numbers of central tandem repeats,were isolated by PCR with these primer pairs. With the exception ofTALE15, which has been reported as hssB3.0 (Shiotani et al (2007) J.Bacteriol 189 (8): 3271-9) the other TALE proteins isolated appear asnovel proteins, as they have not been reported in the public literature.These include TALE13, TALE9, and TALE16, with 13, 9, and 16 TALErepeats, respectively.

The domain map of TALE13 (with the length of the N-cap inferred) isshown in FIG. 1A and the sequence indicating the domains and the aminoacids that determine the DNA sequence that the protein interacts withare indicated in FIG. 1B, along with indicators of the positionalnumbering system used in this work.

Example 2 Truncation of TALE13 and Other TALEs and Effects on DNABinding

As an initial investigation of the range of capping sequences thatprovide maximal activity, several truncations of the TALE were made.These truncations are shown below in Table 4.

TABLE 4 TALE truncation characteristics N Nuclear Clone N Term TermLeucine local- num- +288 to +137 to Repeat rich ization Acidic ber +138+37 R0 units R1/2 region domain domain #1 (−) (+) (+) (+) (+) (+) (+)(+) #2 (−) (+) (+) (+) (+) (+) (−) (−) #3 (−) (+) (+) (+) (+) (−) (−)(−) #4 (−) (−) (+) (+) (+) (+) (−) (−) #5 (−) (−) (−) (+) (+) (−) (−)(−) #6 (−) (+) (+) (+) (+) (+) (−) (−) #7 (−) (+/−) (+) (+) (+) (+) (−)(−) Note: (+) indicates the presence of the region while (−) indicatesits absence

The regions of the truncations are numbered as follows: On theN-terminus, the end point is represented by a number that enumerates thenumber of amino acid residues in an N-terminal direction from the firstbase of the first true TALE repeat (see FIG. 1B). For example, a labelof N+91 describes a truncation on the N-terminus that leaves intact the91 amino acids in the N-terminal direction from the N-terminus of thefirst true repeat. On the C terminus, the end point is represented bythe number of amino acids in the C-terminal direction from the lastamino acid of the last full TALE repeat. Truncation #1, termed TALE-13,clone #1, has the N-terminal 152 amino acids of the full length TALEprotein removed and a single methionine residue added to the resulting Nterminus and thus has an N+137 endpoint (N-cap), making this cloneapproximately 2.5 kb in length. Truncation #2, also has the N-terminal152 amino acids of the full length TALE protein removed, and a singlemethionine residue added to the resulting N-terminus and thus has anN+137 endpoint, as well as the C-terminal sequences downstream of the 5′edge of the NLS, making this clone approximately 2.0 kb in length.Truncation #3 is similar to clone #2 except that it has the leucine-richregion deleted (the leucine-rich region is C-terminal to the half-repeatand extends to C+52 of the C-cap), making this clone approximately 1.6kb in length. Truncation #4 is similar to clone #2 except that on theN-terminus, it has been deleted all the way up and including the R0repeat sequence, making this clone approximately 1.6 kb in length.Truncation #5 is similar to clone #4 except that its deletion on theC-terminal side includes the leucine-rich sequence (similar to clone#2), making this clone approximately 1.4 kb in length. The deducedtarget sequence of the full length TALE 13 protein is TATAAATACCTTCT(SEQ ID NO:54), although there has not yet been an endogenous targetsite identified for this protein. Truncation #6 has 152 amino acidsdeleted from the N-terminus and in the C-terminal regions is similar toclone #2 except that 43 additional amino acids have been deleted.Truncation #7 has 165 amino acids deleted from the N-terminus and hasthe same C-terminal deletion as clone #6. Truncations #6 and #7 arediscussed below.

A standard SELEX assay was run on the truncated TALE proteins toidentify the DNA sequence these proteins bind to (for SELEX methodology,see Perez, E. E. et al. Nature Biotech. 26, 808-816 (2008)), and theresults are presented in Tables 5 and 6. The experiment presented inTable 5 was performed with target library N18TA. The N18TA libraryincludes a DNA duplex with sequence:

N18TA:5′CAGGGATCCATGCACTGTACGTTTNNNNNNNNNNNNNNNNNNAAACCACTTGACTGCGGATCCTGG 3′(SEQ ID NO:55), where N indicates a mixture of all four bases.Additional libraries (as indicated) include the following sequences:

N22AT: (SEQ ID NO: 59)5′CAGGGATCCATGCACTGTACGAAANNNNNNNNNNNNNNNNNNNNNNTTTCCACTTGACTGCGGATCCTGG 3′ N21TA: (SEQ ID NO: 60)5′CAGGGATCCATGCACTGTACGTTTNNNNNNNNNNNNNNNNNNNNNAAACCACTTGACTGCGGATCCTGG3′ N23TA: (SEQ ID NO: 61)5′CAGGGATCCATGCACTGTACGTTTNNNNNNNNNNNNNNNNNNNNNNNAAACCACTTGACTGCGGATCCTGG 3′ N26:5′CAGGGATCCATGCACTGTACGTTNNNNNNNNNNNNNNNNNNNNNNNNNNAACCACTTGACTGCGGATCCTGG 3′ N30CG: (SEQ ID NO: 62)5′CAGGGATCCATGCACTGTACGCCCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGGGCCACTTGACTGCGGATCCTGG 3′

The data is presented below in Table 5 as a base frequency matrix. Ateach position in these matrices, the box indicates the expected RVDtarget base; numbers indicate the relative frequency of each recoveredbase type where 1.0 indicated 100%.

TABLE 5 SELEX results with TALE 13, clone #1

The TALE 13 clone #1 protein appears to be highly selective in itsbinding despite lacking the N-terminal 152 amino acids. The SELEX datafor TALE 13, clone #2 is presented in Table 6. In this figure, the SELEXwas repeated with two different libraries of target sequences, and gavesimilar results with both libraries.

TABLE 6 SELEX Results with TALE 13, clone #2

When clones #3, 4 and 5 were subjected to the SELEX procedure, noconsensus sequences were detected. Thus it appears that the TALE bindingdomains require N- and C-terminal cap sequences comprised in from clone#2 to yield a consensus sequence in this assay. Additional truncationswere made and tested for activity using a DNA binding ELISA assayessentially as described in Bartsevich et al., Stem Cells. 2003;21:632-7. The truncations are presented below in Table 7, which alsoincludes the ELISA results. The starting N-terminus in these truncationsis at amino acid 152, identical to the N-terminus in the #1, #2, and #3truncations discussed above. In this fine-scale truncation series, theend points are as follows.

TABLE 7 ELISA results on fine truncations of TALE13 ELISA results(relative N-Cap C-Cap fluorescence units) N + 137 C + 52 56, 32 N + 121C + 52 8, 9 N + 111 C + 52 10, 12 N + 100 C + 52 8, 9 N + 91  C + 52 9,10 N + 137 C + 95 131, 82, 44 N + 100  C + 115 10, 14 N + 91   C + 11512, 13 N + 0   C + 278 10 N + 0  C + 95 9 N + 0  C + 27 8 N + 137  C +278 12 N + 137 C + 27 10

These data suggest that the efficient TALE binding in this in vitroassay requires residues from between N+122 and N+137 and also frombetween C+53 and C+95 (N-cap residues up to and including N+121 were notsufficient for robust binding and C-cap residues up to and includingC+52 were not sufficient for robust binding).

The preliminary mapping studies allowed the estimation of the minimalN-cap and C-cap sequences of the Xanthomonas TALE to achieve optimalbinding activity. For the N-terminal cap, it appears that the sequencecomprising some number of amino acids between the N+122 and N+137 aminoacids prior to the beginning to the first true repeat are required forDNA binding activity. Similar cap examples for the Ralstonia caps can bemade based on structural homology to the Xanthomonas TALEs (see below inTable 8). In the C-terminal caps, the bold amino acids indicate theRVDs.

TABLE 8 Cap examples Terminus Position Sequence Xanthomonas N-term N+137MVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLN (SEQ ID NO: 363) N-term N+121IKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGV TAVEAVHAWRNALTGAPLN(SEQ ID NO: 364) C-term C+52LTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNR (SEQ ID NO: 365) C-term C+31LTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHL (SEQ ID NO: 366) RalstoniaBased on: N-term YP_002253357.1LKQESLAEVAKYHATLAGQGFTHADICRISRRWQSLRVVANNYPELMAALPRLTTAQIVDIARQRSGDLALQALLPVAAALTAAPLGLSASQIATVAQYGE RPAIQALYRLRRKLTRAPLG(SEQ ID NO: 367) C-term YP_002253357.1LSIAQVIAIACIGGRQALTAIEMHMLALRAAPYNLSPERV (SEQ ID NO: 368)

Example 3 Binding Specificity for Natural TALE Proteins 9 and 16

Two additional natural TALE proteins were subjected to the SELEXprocedure to identify the target DNA sequences that these proteins bind.TALE 9 has 8.5 TALE repeats that specify the following DNA target:TANAAACCTT (SEQ ID NO:56), while TALE16 has 15.5 TALE repeats thatpredict the following target: TACACATCTTTAACACT (SEQ ID NO:57). The dataare presented in Tables 9 and 10. In Table 9, the TALE 9 protein in theclone #2 configurations was used and the results are shown. As with TALE13 clone #2, this experiment was repeated with a second partiallyrandomized DNA library and gave similar data as the first library. Asdescribed above for TALE 13, TALE 9 is highly specific for its targetsequence.

TABLE 9 SELEX results with TALE 9, clone #2

Table 10 shows the SELEX data for the TALE 16 protein with the N18TAlibrary and again demonstrates a high degree of sequence specificity forthe target identified.

TABLE 10 SELEX results with TALE 16, clone #2

Additional truncations were made in the TALE proteins to furtherinvestigate the conditions for efficient DNA binding. Table 4 abovedepicts these truncations. When TALE 9 was tested in the clone #6truncation (Table 11) the DNA binding specificity was maintained(compare Table 11 with Table 9).

TABLE 11 SELEX results with TALE 9, clone #6

Example 4 Reporter Gene Activation by TALE-Fusion Proteins in MammalianCells

To investigate the functional activity of the TALE domain fusions inmammalian cells, engineered reporter constructs were made as follows.One or more copies of the target sequences for the cloned TALE 13 orTALE 15 were inserted in a reporter construct between the NheI and BglII sites thereby placing the targets upstream from the fireflyluciferase expression unit driven by the minimal SV40 promoter in thepGL3 plasmid (Promega) (see FIG. 2). The promoter region of the pGL3plasmid is shown in FIG. 2A and the sequence containing the twopredicted target sites for TALE13 is shown in FIG. 2B. In the experimentdepicted in FIG. 3, the TALE protein construct, together with thereporter plasmid containing 2 targets (FIG. 3A), and an expressionconstruct containing Renilla luciferase (Promega) as an internalcontrol, were co-transfected into human 293 cells. The fireflyluciferase activity induced by each TALE protein was then analyzed 2days after transfection. In response to multiple targets, TALE VP16fusions can synergistically activate the reporter gene expression inmammalian cells (FIG. 3). Additionally, as shown in FIG. 4B, TALEproteins with addition of the VP16 activation domain (TR13-VP16 andTR15-VP16) activate the luciferase reporter gene. Expression of thenatural TALE protein without the VP16 domain does not activateluciferase (TR13 and TR15). Thus the reporter gene activation isobserved only when the correct targets are matched with theircorresponding TALE fusions, suggesting that the transcriptionalactivation results from targeted DNA binding.

Next, the TALE target sequences were inserted in both distal andproximal locations relative to the targeted promoter. In thisexperiment, the TALE13 target was used as shown in FIG. 5A where fourtarget sequences were inserted either upstream (for example “R13x4”) ordownstream (“R13x4D”) of the promoter. The results, shown in FIG. 5Bdemonstrate that optimal activation is seen when the TALE13 bindingsites were placed upstream in close proximity to the promoter ofinterest.

Example 5 Construction of an Artificial TALE Transcription Factor

Having demonstrated that TALE proteins can be linked to atranscriptional regulatory domain to modulate reporter gene expressionin mammalian cells, experiments were performed to engineer TALEtranscription factors with desired targeting specificities. Silentmutations (i.e. a change in the nucleotide sequence without analteration of amino acid sequence) of TR13 VP16 were introduced tocreate two unique restriction sites, ApaI and HpaI, at the beginning ofthe first tandem repeat and the end of last tandem repeat, respectively.These ApaI and HpaI sites were then used for cloning the synthetictandem repeats into the TR13 VP16 backbone to generate the engineeredTALEs with complete N- and C-terminal sequences flanking the tandemrepeats, as well as the VP16 activation domain.

The targeted sequence was GGAGCCATCTGGCCGGGT (SEQ ID NO:58) locatedwithin the NT3 promoter sequence. Previously a ZFP TF 23570 targeting tothis sequence has shown to activate the endogenous NTF3 gene expression(See co-owned U.S. Provisional Patent application 61/206,770). The 17.5tandem repeats from the TALE AvrBs3 were used as a backbone to engineerTALE18 (also termed “NT-L”) such that the tandem repeats of theengineered TALE18 amino acid sequences were altered to specify theintended target nucleotide. The amino acid sequence of the DNA-bindingdomain from engineered TALE18 is shown below in Table 12, where the RVDsare shown boxed in bold:

TABLE 12 DNA-binding domain of engineered TALE18 (NT-L)Engineered TALE18 17.5 repeats

In addition to the four RVDs used in previous engineering efforts (NI,HD, NN, and NG to target A, C, G and T, respectively) we alsoincorporated the NK RVD in a subset of TALE repeats at positionscorresponding to G nucleotides in the DNA target site as it was observedwith a cognate target site guanine in two naturally occurring proteins(see Moscou et al, ibid). Consistent with earlier experimental studies(see Boch et al, ibid), we found that on average NI, HD, NG showed astrong preference for adenine, cytosine, and thymine respectively and NNshowed a preference for guanine, but can also bind adenine. In contrast,the NK RVD shows a strong preference for guanine, representing apotential improvement for engineered TALE proteins that target sitesincluding at least one guanine.

The DNA sequence coding for the 17.5 tandem repeats of the engineeredTALE18 was then derived from the amino acid sequence and synthesized by84 overlapping oligos, each about 40 nucleotides in length, as follows.First, the whole 1.8 kb DNA sequences were divided into 11 blocks, andoverlapping oligos covering each block was assembled by PCR-basedmethod; the 11 blocks was then fused together into 4 bigger blocks byoverlapping PCR and finally, the 4 blocks were assembled into the fulllength by overlapping PCR using the outmost primer pairs. Thesynthesized tandem repeats was then sequence confirmed and cloned intothe ApaI and HpaI sites of TR13-VP16, as described above, to generatethe expression construct of engineered TALE18 (NT-L) targeting to theNT-3 promoter (R23570V).

The specificity of this engineered protein (termed NT-L) was thendetermined by SELEX, and the results are shown below in Table 13. As canbe seen, the data demonstrate that it is possible to engineer anentirely novel TALE protein to bind to a desired sequence. The SELEXselection was also performed with NT-L in the clone #6 truncation (seeabove) as is also shown below in Table 13 demonstrating that, similar toTALE 9, the specificity of the NT-L is maintained within thistruncation. The SELEX experiment was also performed with NT-L in theclone #7 truncation that showed that DNA binding specificity wasmaintained.

TABLE 13 SELEX results with NT-L, clone #2, #6, and #7

The transcriptional activity of the engineered NT-L proteins was thenanalyzed against a luciferase reporter construct containing two copiesof the target sequence. As shown below in Table 14 and FIG. 6A, theengineered NT-L fusion protein (R23570V), containing the engineered 17.5tandem repeats but otherwise identical to TR13-VP16, is capable ofdriving potent reporter gene activation, whereas the similar constructwith no tandem repeats (R0-VP16) does not activate luciferase. The TALEsequences flanking the full length tandem repeats (N-cap and C-cap) arerequired for the reporter gene activation as the deletion of either theN-terminal or C-terminal sequence flanking the repeats (nR23570S-dNC andnR23570S-dNC, respectively) abolished the transcriptional activity. Theconstruct termed nR23570S-dNC contained the SV40 nuclear localizationsignal (n) and the engineered NT-L repeats (R23570) fused to a singlep65 activation domain (S). This construct contained only the repeats butno N-terminal or C-terminal sequence from TALE (dNC). The constructednR23570SS-dNC was same as described for nR23570S-dNC except that it hadtwo p65 activation domains.

As can be seen from Table 14, the highest level of activation of thereporter was found with the R23570V construct. Note that when the NT-Lrepeats were used in the absence of the N-terminal and C-terminalcapping regions, no activation above background was observed in thisassay (compare nR23570S-dNC to mock).

TABLE 14 Reporter activation of NT-L fusion Construct Fold ActivationnR23570S-dNC 1.96 nR23570SS-dNC 3.77 R23570V 74.46 R0-VP16 1.00 Mock1.48

Next, the constructs were used to target the endogenous NTF3 gene to seeif the engineered fusion protein was capable of activating an endogenousgene in its chromosomal locus in a mammalian cell. In the experiment ofFIG. 6B, the engineered NT-L (R23570V), as well as the controlconstructs (R0-VP16, GFP), were transiently transfected into human 293cells. After 2 days following transfection, the NT-3 expression levelwas analyzed by Taqman analysis. As shown in FIG. 6B, expression ofengineered NT-L (R23570V) lead to a substantial increase in NTF3 mRNAexpression in human 293 cells, whereas expression of control proteins(R0-VP16 or GFP) had no effect on NTF3 expression level. This is thefirst time that a specifically engineered TALE domain fusion protein hasbeen used in a mammalian cell to activate expression of an endogenousgene.

An additional exemplary construct was made to determine if all 278residues of the C-terminal regions flanking the TALE repeat domain wasrequired for activity. This additional construct (+95) contained onlythe first 95 residues of the C-terminal region between the TALE repeatdomain and the VP16 activation domain (i.e. C+95 C-cap). FIG. 7 shows adiagram of these two constructs (the +278 construct was referred to asR23570V in FIG. 6) and the effect of these proteins on NTF3 activationat the mRNA and protein levels. Also shown are the SELEX results for thelonger of these constructs (containing the +278 C-terminal (or fulllength) domain). As can be seen in the figure, both TALE transcriptionfactor constructs are able to up-regulate NTF3 expression at both mRNAand protein levels.

Constructs specific for binding in regions in the VEGF, CCR5 and PEDFgene were also generated. As described above, repeat domains wereengineered to bind to these targets by the methodology described above.Target sites for these proteins are shown below in Example 7. Theproteins contained either 10-repeat or 18-repeat DNA binding domains.

Additionally, a series of truncations were made in the 9.5 repeatNTF3-specific and the 9.5 repeat VEGF-specific TALE DNA binding domains.The truncations were expressed in the TNT Coupled Reticulocyte Lysatesystem (Promega) and the lysate was used to bind to the DNA fragments asfollows. The protein were expressed by adding 5 μL of water containing250 nanograms of the nuclease fusion clone plasmid to 20 μL of lysateand incubating at 30° C. for 90 minutes. The binding assays were done asdescribed above. Western blots using standard methodology confirmed thatthe expressed proteins were all equally expressed. The results of thebinding assays are shown in FIG. 8. In these experiments, fortruncations of the N-terminus, the C-terminal amino acid was held atC+95, while for the C-terminal truncations, the N-terminus wasmaintained with the N+137 configuration. As can be seen from the Figure,in this assay, maximal binding was observed when the proteins containedat least 134 amino acids on the N-terminal side of the first truerepeat, and at least 54 amino acids on the C-terminal side of the halfrepeat, and interestingly, this was true for both the TALE DNA bindingdomain targeted to the NTF3 sequence and for the one targeted to theVEGF sequence (compare panels A and B). The truncations around thecritical 134 N-terminal position were repeated using a protein where theC-terminus was truncated to +54 (rather than C+95 as described above)and the C-terminal truncations were repeated where the N-terminus wastruncated to the +134 position (rather than N+137). The data arepresented in FIG. 9 and show a similar drop-off in DNA binding when theC terminus was truncated past +54 and/or when the N terminus wastruncated past +134 as was observed in the previous experiment. Thesedata indicate that the minimal caps for optimal binding in this in vitroaffinity assay extend to positions N+134 and C+54.

Example 6 Dissection of the TALE Functional Domains Involved in DNATargeting in mammalian cells

In this example, various deletions at N-terminal or C-terminal of TALE13proteins, as indicated below in Table 15, were generated.

TABLE 15 TALE 13 deletion constructs Construct Name N-cap C-cap R13 N +137 C + 278 R13-dN N + 8 C + 278 R13-d240N N + 34 C + 278 R13-d223N N +52 C + 278 R13-d145C N + 137 C + 133 R13-d182C N + 137 C + 95 R13-dC N +137 C + 22

All constructs were linked to the VP16 activation domain (constructswith VP 16 were designated “R13V”) and a nuclear localization signal(constructs with NLS were designated “nR13”), and tested for reportergene activation from a reporter construct containing 2 copies ofpredicted TALE13 targets (FIG. 10, top panel).

As shown in FIG. 10, the minimal region that retains robust reporteractivation activity in this set of constructs (see Table 15) isR13V-d182C, which lacks 152 amino acids at its N-terminus and 183 aminoacids at its C-terminus. The result confirms that R0 region precedingthe first tandem repeats and the leucine rich region following the lastrepeat is provides optimal binding in this assay, whereas the regioncontaining nuclear localization signal, and the native activation domainat its C-terminus is dispensable for DNA-targeting in mammalian cells.

Example 7 Demonstration of Nuclease Cleavage Activity of a TALE Linkedto Nuclease Domains

Next, the DNA targeting ability of TALEs in the context of artificialTALE nucleases (TALENs) was evaluated. The DNA targeting domain ofTALE13 as defined in Example 6 was linked to nuclease domains togenerate a construct named as R13d182C-scFokI, which is the same asR13V-d182C described above, except that two copies of the FokI nucleasedomain, linked by 12 copies of GGGS sequence between the FokI domains,were used to replace the VP16 activation domain. The TALEN construct wasthen tested for nuclease activity in a single stranded annealing (SSA)based reporter assay (see co-owned US Patent Publication No.20110014616).

The reporter construct (FIG. 11A, SSA-R13) used in this assay containsthe predicted TALE 13 target, sandwiched by the N-terminal (GF) andC-terminal part (FP) of the GFP coding sequence. The reporter SSA-R13 byitself cannot drive the GFP expression, but the cleavage at the TALE13target will promote homologous recombination (HR) among the N- andC-terminal part of GFP to form a functional GFP transgene. In theexperiment whose results are depicted in FIG. 11B, the SSA-R13 reporterconstruct, together with or without (mock) the TALEN construct, wastransiently nucleofected into K562 cells as described previously.

Two days following nucleofection, the percentage of GFP positive cellswas analyzed by flow cytometry. As shown in FIG. 11B, about 7% GFPpositive cells were generated from SSA-R13 reporter plasmid by the TALENfusions (R13d182C-scFokI), compared to about 1.4% in the controlexperiment lacking the TALE plasmid (mock), representing a significantincrease in the cleavage at TALE13 target in the SSA-R13 reporter.

These data demonstrate that TALE DNA binding domains can be used togenerate functional TALENs for site specific cleavage of DNA inmammalian cells.

TALE domain fusions were also constructed using Fold cleavage halfdomains. For these examples, wild type FokI half cleavage domains wereused so that for nuclease activity, a homodimer must be formed from twoof the fusions. For these fusions, the TALE13 DNA binding domain wasfused to each FokI half domain by cloning the TALE DNA binding domaininto a plasmid adjacent to FokI-specifying sequence. In addition,various linkers were tested for use between the DNA binding domain andthe nuclease domain. Linkers L2 and L8 were used which are as follows:L2=GS (SEQ ID NO:71) and L8=GGSGGSGS (SEQ ID NO:72). The target siteswere cloned into a TOPO2.1 target vector (Invitrogen) with varying gapspacings between each target binding site such that the two wereseparated from each other by 2 to 22 bp. PCR amplification of anapproximately 1 kb region of the target vector was done to generate thetarget DNAs. The TALE DNA binding domains were also truncated asdescribed previously, and are described using the same nomenclature asdescribed above in Examples 2 and 6. The TALE domain nuclease fusionclones were expressed in the TNT Rabbit Reticulocyte Lysate system byadding 5 μL of water containing 250 nanograms of the nuclease fusionclone plasmid to 20 μL of lysate and incubating at 30° C. for 90minutes.

The lysate was then used to cleave the target DNAs as follows: 2.5 μL oflysate were added to a 50 μL reaction containing 50 nanograms ofPCR-amplified target DNA and a final Buffer 2 (New England Biolabs)concentration of 1×. The cleavage reaction was for one hour at 37° C.,followed by a 20 minute heat inactivation stage at 65° C. The reactionwas then centrifuged at high speed to separate the target DNA from thelysate, causing the lysate to condense into a pellet in the reactionwell. The DNA-containing supernatant was pipetted off and run on anethidium bromide-stained agarose gel (Invitrogen) to separate intacttarget DNA from cleaved target DNA. The agarose gel was then analyzedusing AlphaEaseFC (Alpha Innotech) software to measure the amount oftarget DNA present in the large uncleaved DNA band and the two smallerDNA bands resulting from a single cleavage event of the target DNA. Thepercentage of cleaved DNA out of the total amount of target DNA loadedinto the gel represents the percent cleavage in each reaction.

We desired to minimize the flanking regions of the TALE proteins in aneffort to pare the fusions down to the specific regions required forefficient binding, reasoning that trimming the extraneous peptidesequence would provide a more constrained attachment of the FokIcleavage domain, which could improve the catalytic activity of theTALENs. The truncations made on the N- and C-terminal ends (SEQ ID NO:73and SEQ ID NO:369) of the TALE DNA binding domain were made as shownbelow where the truncation sites are indicated above the amino acidsequence, and the predicted secondary structure (C=random coil, H=helix)is indicated underneath the sequence:

C-Cap:                                               C+28       C+39       C+50    C+58LTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACL G GRPALDAVKK GLPHAPALIKR T NRRIPER T SHRVCCHHHEEEEECCCCCHHHHHHHHHCCCCCCHHHHHCCHHHHHHHHHCCCCHHHHHHHCCCCCCHHHHHHHCCCCCCCCCCCCC+63            C+79            C+95 A DHAQVVRVLGFFQCH S HPAQAFDDAMTQFGMS RHGL CCCHHHHHHHHHHHCCCCHHHHHHHHHHHHCCCHHHH N-Cap:N+137  N+130      N+119          N+104MVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGV   N+134CCCHHHCCCCHHHHHHHCHHHHHHHHHHHHHHHCCCCCHHHHHEECCCHHHHHHHHCCHHHHHHHCCHHHHHHHHHH

The results of the C-terminal deletion studies are shown in FIGS. 12 and13. FIG. 12 shows the cleavage of the target sequences by visualizingthe cleavage products on ethidium bromide stained agarose gels. In FIG.12, L2 or L8 indicates the linker used, and the number beneath each laneindicated the by gap between the two target DNA binding sites of thedimer. ‘S’ indicates the presence of only one target DNA binding sitesuch that an active nuclease homodimer cannot form on the DNA. “Pml1”indicates the positive control reaction of cleavage using acommercially-available restriction enzyme (New England Biolabs) of aunique restriction site located in the cloned DNA target sequence nextto the TALE binding sites. Cleavage at the PmlI site indicates that thecloned target site exists in the PCR-amplified target DNA and also showsthe approximate expected size of cleaved DNA. Blank indicates thenegative control TNT reaction without the TALEN encoding plasmid suchthat no TALEN was produced. The data is depicted in a graphical formatin FIG. 13, and shows that the cleavage activity of the protein greatlyincreases with C+28 and C+39 C-caps for a spacer length of at least 9bases. These experiments were continued and further C-caps (C-2, C+5,C+11, C+17, C+22, C+25, C+28 and C+63) were constructed. The results aresummarized below in Table 16. “Spacer” indicates the number of basepairs between the target sites and “SC” indicates those samplescontaining only one binding site in the target.

TABLE 16 C terminal truncations of TALE13-homodimer pairings in vitrospacer C−2 C+5 C+11 C+17 C+22 C+25 C+28 C+63 SC  0.0%  0.0%  0.0% 11.4%19.5%  7.7%  8.0% 10.4%  4 12.9% 11.9% 17.4% 27.9% 51.8% 21.1% 19.4%26.6%  8 10.2% 23.4% 27.4% 33.6% 46.2% 26.1% 35.4% 15.2% 10 16.9% 97.7%98.9% 98.6% 99.9% 93.4% 94.8% 12.8% 12  1.1% 99.3% 98.5% 97.8% 98.1%96.5% 96.5% 27.1% 14  5.1% 98.7% 96.9% 99.0% 98.5% 98.5% 96.2% 32.6% 16 1.4% 98.3% 98.9% 99.9% 97.6% 97.5% 96.1% 37.1% 20  4.9% 99.2% 98.9%99.9% 98.8% 99.3% 98.3% 28.9%

As can be seen from the data presented above, it appears that theproteins become less active in this assay as fusion nucleases when theC-terminus is truncated past approximately C+5.

Cleavage activity of TALE13 nucleases with additional C-terminaltruncation points when presented with a target with the indicated spacerwas also assessed and results are shown in Table 17 below. “S” indicatesthat the cleavage target contained a single binding site for TALE13.

TABLE 17 TALE 13 nuclease C-terminal truncations C-terminal truncationpoint Spacer (bp) C+28 C+39 C+50 C+63 C+79 C+95  2 <5 <5 <5 <5 <5 <5  4<5 <5 <5 <5 <5 <5  6 <5 <5 <5 <5 <5 <5  8 <5 <5 <5 <5 <5 <5 10 96 45 <5<5 <5 <5 12 100 99 62 33 26 <5 14 100 100 82 70 52 <5 16 100 100 83 7056 <5 18 99 100 81 75 59 <5 20 89 99 93 75 65 <5 22 99 99 94 79 69 <5 24100 100 92 83 60 <5 S >5 <5 <5 <5 <5 <5

Similar to the work done on the C-terminal region of the TALE proteins,deletions were made in the N-terminus as well. The data is presented inFIG. 14 and it is apparent that the activity of the protein with theN-terminal deletions is diminished when truncations are introducedrelatively close to the N+137 position. In this Figure, each column islabeled with the corresponding N-terminal truncation and the number ofthe separate clones that were used. “S” indicates that only a singlebinding site was present in the target. The sum of these resultsindicates that the TALENs can be quite active when linked to either FokIhalf domains or to two half domains which can interact in a single chainconfiguration, but the length of the N-cap and C-cap has an effect onthe DNA cleavage properties of the resulting TALENs.

TALENs were constructed to bind to an endogenous target in a mammaliancell. The 10 repeat NTF3 binding domain was linked to a FokI half domainas described above. In addition, a NTF3 specific partner (rNTF3) wasconstructed commercially using standard overlapping oligonucleotideconstruction technology. The synthetic NTF3 partner was made with threevariants at the C terminus: C+63, C+39 and C+28, and the TALE DNAbinding domain was cloned into a standard ZFN vector which appends anepitope tag and a nuclear localization signal to the C-terminus and thewild-type FokI cleavage domain to the C-terminus. The complete aminoacid sequences of the constructs used in these experiments are shown inExample 23.

In addition to the 9.5 repeat NTF3-FokI fusion, and the 18 repeatNTF3-specific NT-L protein, TALENs were also made to target a sitespecific for the VEGF A gene. This fusion protein contained 9.5 repeatunits and was constructed as described above. The 18 repeat NT-L and theVEGF-specific TALENs were also made with either a C terminal truncationof either +28, +39 or +63. These synthetic fusion nucleases were thenused in vitro in nuclease assays as above, in various combinations. Thesubstrate sequences are shown below with the capital letters indicatingthe target binding sites for the various fusions:

NTF3-NTF3 substrate (SEQ ID NO: 77):              NT3-18/NT3-10 gcacgtggcGGAGCCATCTGGCCGGGTtggctggttataaccgcgcagattctgttcaccgcgcgataacgtgcaccgcctcggtagaccggcccaaccgaccaatatTGGCGCGTCTAAGACAAGtggcgcgcta                                              rNT3 NT3-VEGF substrate(SEQ ID NO: 78):              NT3-18/NT3-10 gcacgtggcGGAGCCATCTGGCCGGGTtggctggttatgaagggggaggatcgatcggacgcgcgataacgtgcaccgcctcggtagaccggcccaaccgaccaatacTTCCCCCTCCtagctagcctgcgcgcta                                               VEGF-10 VEGF-NT3substrate (SEQ ID NO: 79):                    VEGF-10 gcacgtggccatggactCCTCCCCCTTcagctggttataaccgcgcagattctgttcaccgcgcgataacgtgcaccggtacctgaggagggggaagtcgaccaatatTGGCGCGTCTAAGACAAGtggcgcgcta                                          NT3-18/NT3-10

The results from these studies are presented below in Tables 18 andTable 19.

TABLE 18 TALEN pairs specific to human NTF3 In vitro cleavage SamplesLeft NT3 Right rNTF3 (av) 1 16 R10 C28L2 C28L2 20% 2 17 R10C28L2 C39L226% 3 18 R10 C28L2 C63L2 42% 4 19 R10 C39L2 C28L2 51% 5 20 R10 C39L2C39L2 43% 6 21 R10 C39L2 C63L2 60% 7 22 R10 C63L2 C28L2 66% 8 23 R10C63L2 C39L2 57% 9 24 R10 C63L2 C63L2 36% 10 25 R18 C28L8 C28L2 16% 11 26R18 C28L8 C39L2 15% 12 27 R18 C28L8 C63L2 11% 13 28 R18 C63L2 C28L2  6%14 29 R18 C63L2 C39L2  4% 15 30 R18 C63L2 C63L2  2%Note that Table 18 shows duplicate testing of each TALEN pair. Forexample, samples 1 and 16 are the same combination of TALEN monomers.

TABLE 19 TALENs targeted to combinations of either NTF3/NTF3 orNTF3/VEGF Pairs 1 protein control NT-L NN site NT3 R10, NT3 R10, NT3R10, VEGFR10, rNT3 C48 L2 #1 4.7% C+28 L2 #1 C+28 L2 #2 C+63 C+28 L2rNT3 C28 L2 #2 4.3% NT-R rNT3 C28 L2 #1 38.4% 46.4% 72.7% 41.8% rNT3 C39L2 #1  3.1% rNT3 C28 L2 #2 27.4% 27.9% 69.7% 27.6% rNT3 C39 L2 #2  2.3%rNT3 C39 L2 #1 41.1% 42.1% 62.0% 37.8% rNT3 C+63 L2  2.5% rNT3 C39 L2 #232.3% 33.3% 62.4% 32.5% NT3 R10 C28 L2 #1  3.5% rNT3 C63R #1 12.6% 10.7% 4.4%  3.4% NT3 R10 C28 L2 #2 14.6% rNT3 C63 #2 63.3% 59.6% 38.4% 61.8%NT3 R10 C63  4.1% VegF R10 C28 L2 90.0% 95.0% 90.8% VegF R10 C63 94.1%96.5% 72.7% NN refers to the relevant portion of the endogenous NTF3target with a binding for both the left (NT-L) and the right (NT-R) NTF3TALENs. #1 or #2 refers to different clones of the same construct.

Thus, these proteins are active as nucleases in vitro.

These proteins were also used in an assay of endonuclease activity in amammalian cell using the SSA reporter system described above. A targetsubstrate (shown in FIG. 15A, SEQ ID NO:452) was cloned in between thedisjointed GFP reporter such that cleavage at the NTF3 site followed byresection will result in a whole GFP reporter capable of expression.This substrate contains both a NTF3 target sequence and a targetsequence specific for targeting the CCR5 gene. FIG. 15B depicts theresults of this experiment using a selection of the NTF3-specific TALEproteins. In this experiment the following NTF3-specific TALEN fusionswere used. TALE13C28L2 is the TALE13 derivative described above with aC+28 truncation and the L2 linker. rNT3R17C28L2 is the 17.5 repeatNT3-specific protein (that targets the reverse strand of the DNA withrespect to the coding strand of the NT3 gene) with the C+28 truncationand L2 linker. rNT3R17C39L2 is the similar construct with the C+39 Cterminus, and rNT3R17C63L2 has the C+63 C terminus. This rNT3R17 DNAbinding domain is also termed NT-R. The 8267EL/8196zKK is a controlusing a pair of CCR5 specific zinc finger nucleases. The data labeled as“−NT3 R18 C28L8” depicts the results in the absence of the NTF3 specificpartner (that targets the forward strand of DNA with respect to thecoding strand of the NTF3 gene), while the data labeled as “+NT3 R18C28L8” depicts the results in the presence of the partner. In this case,the partner is an NTF3 specific protein with 17.5 repeats, truncated atthe C28 position and containing the L8 linker. As can be seen in theFigure, the correct pairing of the TALENs leads to efficient cleavage ofthe reporter gene and thus reporter gene expression.

Example 8 Use of Engineered TALENs to Cleave an Endogenous Locus in aMammalian Cell

The dimer pairs described above that were targeted to the NTF3 locus(see Table 18) were then tested at the endogenous locus in a mammaliancell. Dimer pairs as shown were nucleofected into K562 cells using theAmaxa Biosystems device (Cologne, Germany) with standards methods assupplied by the manufacturer and subjected to a transitory cold shockgrowth condition following transfection (see U.S. application Ser. No.12/800,599).

Cells were incubated at 30° C. for three days and then the DNA isolatedand used for Cel-I analysis. This assay is designed to detect mismatchesin a sample as compared to the wild type sequence. The mismatches are aresult of a double strand break in the DNA due to cleavage by the TALENthat are healed by the error prone process of non-homologous end joining(NHEJ). NHEJ often introduces small additions or deletions and the Cel-Iassay is designed to detect those changes. Assays were done asdescribed, for example, in U.S. Patent Publication Nos. 20080015164;20080131962 and 20080159996, using the products amplified with thefollowing primers: LZNT3-F4: 5′-GAAGGGGTTAAGGCGCTGAG-3′ (SEQ ID NO:80)and LZNT3-1077R: 5′-AGGGACGTCGACATGAAGAG-3′ (SEQ ID NO:81). Theseprimers amplify a 272 bp amplicon from the endogenous sequence, andcleavage by the Cel-I assay will produce products of approximately 226and 46 bp. While the 226 bp products are visible, the 46 bp products aredifficult to see on the gel due to their size. The results are shown inFIG. 16 where the percent genome modification observed is indicated inthe lanes that include the Cel-I enzyme. As is evident from the Figure,there are nuclease-induced mutations occurring in these samples, and thesamples are reproducible in duplicate (e.g. compare lanes 7 and 22, orlanes 12 and 27).

The studies were repeated with pairs 15, 13, 12, and 10 (see Table 18),using cells that were incubated at either a 37° C. or 30° C. aftertransfection, and the results are shown in FIG. 17. First, the NT-R TALEDNA binding domain was tested in the SELEX assay as previously describedand the results are shown in FIG. 17A. When expressed in K562 cells,these proteins yielded robust gene modification as revealed by the Cel-Iassay, with estimated levels of 3% and 9% for the most activeheterodimer (pair 12) tested at 37° C. and 30° C. (see FIG. 17B).Moreover Sanger sequencing identified 7 mutated alleles out of 84analyzed in the 30° C. sample and also revealed a mutation spectrum(minor deletions) consistent with error-prone break repair vianon-homologous end joining (NHEJ) (FIG. 17C).

These studies show that TALEN architecture as described herein can driveefficient NHEJ-mediated gene modification at an endogenous locus and ina mammalian cell.

These studies also reveal compositions that may be used to link anuclease domain to a TALE repeat array that provides highly activenuclease function. The samples were also subjected to deep sequencing atthe NTF3 locus. Samples were barcoded with a 4 bp sequence and a 50 bpread length was used on an Illumina Genome Analyzer instrument(Illumina, San Diego Calif.). Sequences were processed with a custompython script. Sequences were analyzed for the presence of additions ordeletions (“indels”) as hallmarks of non-homologous end joining (NHEJ)activity as a result of a double stranded break induced by nucleaseactivity. The results are presented in FIG. 18. In the endogenous locus,there is a 12 base pair gap between the target sequences recognized bythese two proteins (see FIG. 18A). As shown in FIG. 18B, there arenumerous indels that demonstrate activity against the endogenous NTF3locus in a mammalian cell. In FIG. 18B, the wild type sequence at theendogenous locus is indicated by “wt”.

Example 9 Targeted Integration into an Endogenous Locus Following TALENCleavage

TALE-mediated targeted integration at NTF3 could happen via the HDR DNArepair pathway or via the NHEJ pathway. We designed an experiment toassay TALE-mediated targeted integration at NTF3 based on the capture ofa small double-stranded oligonucleotide by NHEJ. We have previouslyshown capture of oligonucleotides at the site of ZFN-induced DNAdouble-strand breaks (DSBs). This type of targeted integration wasenhanced by (but did not absolutely require) the presence of 5′overhangs complementary to those created by the FokI portions of the ZFNpair. Fold naturally creates 4 bp 5′ overhangs; in the context of a ZFN,the FokI nuclease domain creates either 4 bp or 5 bp 5′ overhangs. Sincethe position and composition of the overhangs left by NTF3 TALENs isunknown, we designed nine double-stranded oligonucleotide donors withall possible 4 bp 5′ overhangs in the 12 bp spacer region between theNTF3 TALEN binding sites (NT3-1F to NT3-9R). (see Table 20).

TABLE 20 PCR primers used for Targeted Integration assay PCR band NameSequence size NT3-1F 5′ T*G *GCGTACGGATCCAAGCTTCGTCGACCTAGCC 3′ (SEQ IDNO: 82) 461 bp NT3-1R 5′ G*C *CAGGCTAGGTCGACGAAGCTTGGATCCGTAC 3′ (SEQ IDNO: 83) NT3-2F 5′ G*G *CTGTACGGATCCAAGCTTCGTCGACCTAGCC 3′ (SEQ ID NO:84) 462 bp NT3-2R 5′ A*G *CCGGCTAGGTCGACGAAGCTTGGATCCGTAC 3′ (SEQ ID NO:85) NT3-3F 5′ G*C*TGGTACGGATCCAAGCTTCGTCGACCTAGCC 3′ (SEQ ID NO: 86) 463bp NT3-3R 5′ C*A*GCGGCTAGGTCGACGAAGCTTGGATCCGTAC 3′ (SEQ ID NO: 87)NT3-4F 5′ C*T*GGGTACGGATCCAAGCTTCGTCGACCTAGCC 3′ (SEQ ID NO: 88) 464 bpNT3-4R 5′ C*C*AGGGCTAGGTCGACGAAGCTTGGATCCGTAC 3′ (SEQ ID NO: 89) NT3-5F5′ T*G*GTGTACGGATCCAAGCTTCGTCGACCTAGCC 3′ (SEQ ID NO: 90) 465 bp NT3-5R5′ A*C*CAGGCTAGGTCGACGAAGCTTGGATCCGTAC 3′ (SEQ ID NO: 91) NT3-6F5′ G*G*TTGTACGGATCCAAGCTTCGTCGACCTAGCC 3′ (SEQ ID NO: 92) 466 bp NT3-6R5′ A*A*CCGGCTAGGTCGACGAAGCTTGGATCCGTAC 3′ (SEQ ID NO: 93) NT3-7F5′ G*T*TAGTACGGATCCAAGCTTCGTCGACCTAGCC 3′ (SEQ ID NO: 94) 467 bp NT3-7R5′ T*A*ACGGCTAGGTCGACGAAGCTTGGATCCGTAC 3′ (SEQ ID NO: 95) NT3-8F5′ T*T*ATGTACGGATCCAAGCTTCGTCGACCTAGCC 3′ (SEQ ID NO: 96) 468 bp NT3-8R5′ A*T*AAGGCTAGGTCGACGAAGCTTGGATCCGTAC 3′ (SEQ ID NO: 97) NT3-9F5′ T*A*TAGTACGGATCCAAGCTTCGTCGACCTAGCC 3′ (SEQ ID NO: 98) 469 bp NT3-9R5′ T*A*TAGGCTAGGTCGACGAAGCTTGGATCCGTAC 3′ (SEQ ID NO: 99) Internal F5′ GGATCCAAGCTTCGTCGACCT 3′ (SEQ ID NO: 100) GJC 273R5′ CAGCGCAAACTTTGGGGAAG 3′ (SEQ ID NO: 101) Note *in the primer sequenceindicates the two 5′ terminal phosphorothioate linkages. All primerslack 5′ phosphates.

These donors contain two 5′ terminal phosphorothioate linkages and lack5′ phosphates, and a binding site for the primer Internal F.Complementary oligonucleotides (NT3-1F with NT3-1R, e.g.) were annealedin 10 mM Tris pH 8.0, 1 mM EDTA, 50 mM NaCl by heating to 95° andcooling at 0.1°/min to room temperature. Donor oligonucleotides (5 μL of40 μM annealed oligonucleotide) were individually transfected with eachof eight different TALEN pairs (A-H, 400 ng each plasmid, see Table 21)in a 20 μL transfection mix into 200,000 K562 cells using an AmaxaNucleofector (Lonza) set to program FF-120 and using solution SF.

TABLE 21 NT3-specific TALEN pairs Pair TALEN 1 TALEN 2 A NT3 R10 C28rNT3 C39 B NT3 R10 C28 rNT3 C63 C NT3 R10 C39 rNT3 C28 D NT3 R10 C39rNT3 C39 E NT3 R10 C39 rNT3 C63 F NT3 R18 C28 rNT3 C28 G NT3 R18 C28rNT3 C39 H NT3 R18 C28 rNT3 C63

Cells were harvested three days post-transfection and lysed in 50 μLQuickExtract solution (Epicentre). One microliter of the crude lysatewas used for PCR analysis as described below.

We assayed targeted integration of the oligonucleotide donor into theDSB created by the NTF3 TALEN by PCR amplification of the junctioncreated by the oligonucleotide and the chromosome using the Internal Fand GJC 273R primers. The expected size of the PCR amplicon based onperfect ligation of the oligonucleotide donor varies depending on theposition of the break in the chromosome. As can be seen in FIG. 19,integration of the donor was detected with many combinations of TALENand donor overhangs. Maximal signal was seen with the CTGG and TGGToverhangs near the center of the 12 bp spacer region. Endogenouschromosomal loci containing donors captured by NHEJ were sequenced andare shown in FIG. 20. The NTF3 target locus (top duplex) and one of theoligonucleotide duplexes used for this study (bottom duplex) are shownand the binding sites for NT-L+28 and NT-R+63 are underlined in the topsequence. The cleavage overhang that will most efficiently capture theduplex (5′ CTGG) is also highlighted. Also shown in FIG. 20B is a secondoligonucleotide duplex used for this study. Binding sites for NT-L+28and NT-R+63 are underlined in the top sequence. The cleavage overhangthat will most efficiently capture this second duplex (5′ TGGT) is alsoshown. The TALENs NT-L+28 and NT-R+63 were then expressed in K562 cellsin the presence of the oligonucleotide duplex shown in FIG. 20A.Junctions between successfully integrated duplex and genomic DNA werethen amplified using one primer that anneals within the duplex and oneprimer that anneals to the native NTF3 locus. The resulting ampliconswere cloned and sequenced. The “expected” sequence in FIG. 20C indicatesthe sequence that would result from a perfect ligation ofoligonucleotide duplex to the cleaved locus. The box highlights thelocation of the duplex overhang in the junction sequences. The bottomtwo lines provide junction sequences obtained from this study. As shown;eleven junction sequences resulted from perfect ligation of duplex tothe cleavage overhang, while one junction sequence exhibited a shortdeletion (12 bp) consistent with resection prior to repair by NHEJ. FIG.20D shows results from experiments as shown in FIG. 20C except that theoligonucleotide duplex shown in FIG. 20B was used, which has a 4 bpoverhang that is shifted by one base relative to the duplex shown inFIG. 20A. The lowest four lines provide junction sequences obtained fromthis study. As shown, four distinct sequences were identified, whicheach exhibit short deletions consistent with resection prior toNHEJ-mediated repair.

Example 10 Efficient Assembly of Genes that Encode Novel TALE Proteins

The DNA sequence encoding TALE repeats found in natural proteins is asrepetitive as their corresponding amino acid sequence. The natural TALEtypically have only a few base pairs' worth of difference between thesequences of each repeat. Repetitive DNA sequence can make it difficultto efficiently amplify the desired full-length DNA amplicon. This hasbeen shown when attempting to amplify DNA for natural TALE-containingproteins. Further analysis of the DNA sequence of the TALE-repeatprotein above using Mfold (M. Zuker Nucleic Acids Res. 31(13):3406-15,(2003)) revealed that not only do they have repetitive sequencedisrupting efficient amplification, but also that they contain verystable secondary structure. In this analysis, 800 base pairs of sequencewere analyzed starting at the 5′ end of the nucleic acid encoding thefirst full repeat sequence. Thus, the nucleic acid sequence analyzedcontained approximately 7.5 repeat sequences. Several of these secondarystructures are shown in FIG. 21.

These structures can occur between any of the TALE repeats or betweenrepeats that are not adjacent. To provide efficient amplification of DNAsequences containing TALE repeats, introduction of silent mutations todisrupt this secondary structure and bias the reaction towards thefull-length amplicon were made in the regions of the TALE repeats thatserve to stabilize the secondary structure. Primers were then made toallow efficient amplification of the TALE sequence or interest. The PCRamplification product was then sequenced for verification and cloned foruse in fusion proteins. In addition, silent mutations were made in theTALE nucleotide sequence for codon optimization in mammalian cells.Similar codon optimization can be used for optimal expression in otherhost cell systems (e.g. plant, fungal etc.).

Example 1 Method for Rapid Construction of Genes Encoding TALE FusionProteins

To allow for the rapid assembly of a variety of TALE fusion proteins, amethod was developed to create an archive of repeat modules which couldbe linked together to create a TALE DNA binding domain specific fornearly any chosen target DNA sequence. Based on the desired target DNAsequence, one or more modules are picked and are retrieved via a PCRbased approach. The modules are tandemly linked and ligated into avector backbone containing the fusion partner domain of choice.

Modules containing four TALE repeat units were constructed withspecificity for each of the 256 possible DNA tetranucleotide sequence(for example, one module for the AAAA target, one for AAAT etc.). Inaddition, modules were also created for all 64 possible DNAtrinucleotide targets, all possible 64 dinucleotide DNA targets as wellas 4 single nucleotide targets. For the dipeptide recognition region(also referred to as an RVD—Repeat Variable Dipeptide), the followingcode was used: For recognition of Adenine, the RVD was NI(asparagine-isoleucine), for Cytosine, the RVD was HD(histadine-aspartate), for Thymine, the RVD was NG (asparagine-glycine),and for R (comparable specificity for Guanine or Adenine), the RVD wasNN (asparagine-asparagine). In addition, in some engineered TALEs, theRVD NK (asparagine-lysine) was chosen for recognition of G because itappeared to give higher specificity for G than NN in some proteins.Furthermore, the penultimate position N-terminal of the RVD (position 11of the repeat unit) was N or asparagine (typically this position is an Sor serine). This module archive can be expanded by using any other RVDs.

The PCR specificity, cloning and manipulation of DNA bearing perfectsequence repeats is problematic. Thus, in order to construct thearchive, many natural TALE repeat sequences were analyzed to see wherevariability in amino acid sequence could be tolerated in an attempt todiversify repeat sequences at the DNA level. The results are depicted inFIG. 22, where letter size is inversely related to observed diversity ata given position: larger letters indicate less tolerance of diversitywhile smaller letters indicate positions where other amino acids aresometimes observed. For example, at position 1, the first amino acid ofthe repeat unit, an L, or leucine is essentially invariantly observed.However, at position 4, three different amino acids are sometimes found:an E, or glutamate, an A, or alanine, or a D, or aspartate. In addition,the nucleotide sequence encoding the various repeat modules was alsoaltered to exploit the redundancy in the genetic code such that codonsencoding specific amino acids may be interchanged allowing the DNAstrand encoding the repeat unit to have a different sequence fromanother repeat unit, but the amino acid sequence will remain the same.All of these techniques were utilized to pools of modules that could beused to construct engineered TALE DNA binding domains where the interiorof the DNA binding domain could recognize any desired target.

To allow the designer to specify the position of the modules, a type IIS restriction enzyme was used, BsaI, which cleaves to the 3′ end of itsDNA target site. BsaI recognizes the sequence shown below. Alsoillustrated are the “sticky ends” (SEQ ID NOs:102-105) of the cleavedDNA left following enzymatic cleavage:

5′...GGTCTCNNNNNN...3′ 5′...GGTCTCN  NNNNN...3′ 5′...CCAGAGNNNNNN...3′5′...CCAGAGNNNNN  N...3′       Recognition site ->           Aftercleavage

As will be appreciated by the artisan, the sequence of the sticky endsis dependent upon the sequence of the DNA immediately 3′ of therestriction recognition site, and thus the ligation of those sticky endsto each other will only occur if the correct sequences are present. Thiswas exploited to develop PCR primers to amplify the desired modules thatwould have known sticky ends once the PCR amplicons were cleaved withBsaI. The PCR products were then combined following BsaI cleavage toallow ligation of the products together in only the order specified bythe user. An assembly scheme to ligate up to four modules that consistof 1 to 16 full TALE repeats is depicted in FIG. 23. The primers usedwere as follows where the numbering corresponds to that shown in theFigure. While the listed primers are intended to be used to ligate up tofour modules, by using the same concept, more primers can be added inorder to ligate more than four modules.

Primers:

T1F-Bsa (SEQ ID NO: 106) GGATCCGGATGGTCTCAACCTGACCCCAGACCAG T1R-Bsa (SEQID NO: 107) GAGGGATGCGGGTCTCTGAGTCCATGATCCTGGCACAGT T2F-Bsa (SEQ ID NO:108) GGATCCGGATGGGTCTCAACTCACCCCAGACCAGGTA T2R-Bsa (SEQ ID NO: 109)GAGGGATGCGGGTCTCTCAGCCCATGATCCTGGCACAGT T3F-Bsa (SEQ ID NO: 110)GGATCCGGATGGGTCTCAGCTGACCCCAGACCAG T3R-Bsa (SEQ ID NO: 111)GAGGGATGCGGGTCTCTCAAACCATGATCCTGGCACAGT T4F-Bsa (SEQ ID NO: 112)GGATCCGGATGGGTCTCATTTGACCCCAGACCAGGTA T4R-Bsa (SEQ ID NO: 113)CTCGAGGGATGGTCTCCTGTCAGGCCATGATCC

When using this method, the ligation of the BsaI cleaved PCR ampliconscan only occur where the 3′ end of the “A” module ligates to the 5′ endof the “B” module, the 3′end of the “B” module can only ligate to the 5′end of the “C” module etc. In addition, the vector backbone that theligated modules are cloned into also contains specific BsaI cleavedsticky ends, such that only the 5′ end of the “A” module, and only the3′ end of the “D” module will ligate to complete the vector circle.Thus, position of each module within the engineered TALE DNA bindingdomain is determined by the PCR primers chosen by the user.

At the current time, DNA target sites for TALE DNA binding domains aretypically flanked by T nucleotides at the 5′ end of the target (which isrecognized by the R0 repeat) and at the 3′ end of the target (which isrecognized by the R1/2 repeat). Thus, the vector backbone has beendesigned such that the ligated PCR amplicons containing the specifiedmodules are cloned in frame between R0 and R1/2 sequences within thevector. In addition, the vector contains the user specified C-terminaldomain type (truncated or not) of the TALE protein and the exogenousdomain of choice for fusion partner. In the design depicted in FIG. 23,the exogenous domain is a FokI domain, allowing for the production of aTALE nuclease. The vector further contains sequences necessary forexpression of the fusion protein such as a CMV promoter, a nuclearlocalization signal, a tag for monitoring expression, and a poly A site.This vector can now be transfected into a cell of the user's choice. Inaddition, the vector can be further modified to contain selectionmarkers, domains or other genes as desired and/or required for differentcellular systems.

Example 12 Design and Characterization of Specific Endogenous TALENs

To evaluate the TALEN design method, we sought to demonstrate TALENmediated gene modification near the position of the delta 32 mutation(shown below in Bold underline) within the human CCR5 gene (see StephensJ C et al, (1998) Am J Hum Gen 62(6): 1507-15). For this study, wedesignated a cluster of four “left” and four “right” binding sites atthe location of delta 32 (see below), which defined a panel of 16 dimertargets (SEQ ID NO:114-122).

L532        5′CTTCATTACACCT L538          5′TCATTACACCTGCAGCTL540               5′ACACCTGCAGCTCTL543               5′ACACCTGCAGCTCTCAT5′ AAAAAGAAGGTCTTCATTACACCTGCAGCTCTCATTTTCCATACAGTCAGTATCAATTCTGGAAGAATTTCCAGACATT   TTTTTCTTCCAGAAGTAATGTGGACGTCGAGAGTAAAAGGTATGTCAGTCATAGTTAAGACCTTCTTAAAGGTCTGTAA5′ R549                                       TATGTCAGTCATAG 5′R551                                         TGTCAGTCATAGT 5′R557                                               TCATAGTTAAGACCTTC 5′R560                                                  TAGTTAAGACCTTCT 5′

Within this panel, individual targets were separated by a range of gapsizes—from 5-27 bp. TALEN proteins were assembled using the methodsdescribed in Example 11, such that in all proteins described (unlessspecifically noted), the RVD specifying was NG, for ‘A’ was NI, for ‘C’was HD and for ‘G’ was NN. Next, two alternative proteins were generatedfor each target, bearing a C-terminal segment of either 48 or 83residues. Finally, all pairwise combinations of “left” and “right”proteins (8×8=64 total) were expressed in K562 cells and assayed formodification of the endogenous locus. See Table 22 below (day 3 and day10):

TABLE 22 Pairwise combinations of activity for CCR5 Δ32-specific TALENtruncations Day 3 modification levels Right Nuclease +28 +63 R549 R551R557 R560 R549 R551 R557 R560 Left +28 L532 <1 <1 <1 <1 <1 <1 <1 <1Nuclease L538 2% 21%  2%  3% <1 12% 26% 21% L540 <1 <1 <1 <1 <1 <1  5%<1 L543 <1 <1 10% <1 <1 <1 21% 12% +63 L532 <1 <1 <1 <1 15%  8% <1 <1L538 <1  6% 30% 24% <1  5% 27% 21% L540 <1 <1 20% 14% <1 <1 24% 19% L543<1 <1 20%  6% <1 <1 12% 24% Day 10 modification levels Right Nuclease+28 +63 R549 R551 R557 R560 R549 R551 R557 R560 Left +28 L532 NucleaseL538 3% 15%  3% 3% 5% 21% 18% L540  3% L543 11% 20% 11% +63 L532 11% 4%L538  5% 23% 23%  3% 26% 17% L540 12% 9% 28% 13% L543 16% 5% 12% 15%

Since the target sites contained a variety of gap sizes, data concerningthe most active nucleases can also be analyzed with respect to thedistance between the two target sites. Shown below in Table 23 is asimilar panel to those above in Table 22, except that it shows the gapsizes for the target sites.

TABLE 23 Gap sizes for pairwise combinations R549 R551 R557 R560 L532 16bp 18 bp 24 bp* 27 bp* L538 10 bp* 12 bp 18 bp 21 bp L540  8 bp* 10 bp*16 bp 19 bp L543  5 bp*  7 bp* 13 bp 16 bp *indicates pairings wherethere was <1% gene correction activity as assayed by the Cel I assay(compare to Table 22, +63/+63)

Thus, the data from Table 22 and Table 23 can be compared to determinethat the range of gap sizes where these pairs are most active includes12 to 21 bp but excludes gaps of less than 11 bp or more than 23 bp.

To demonstrate that our TALEN architecture could induce gene editing viathe other major cellular DNA repair pathway: homology directed repair(HDR), a second locus within CCR5 (termed locus 162) that had shownpromise in prior studies as a potential safe-harbor for transgeneintegration (see Lombardo et al (2007) Nat Biotechnol 25: 1298-1306) wastargeted. Four “left” and four “right” right binding sites weredesignated (see below, SEQ ID NO:123-131), and two alternative TALENswere constructed for each (the +28 and +63 variants), and the +28/+28and +63/+63 pairings were screened for NHEJ-mediated gene modificationusing the Cel-I assay (SEQ ID NOs:370-379).

L161                    5′GCTGGTCATCCTCATL164                       5′GGTCATCCTCATCCTL167                          5′CATCCTCATCCTGATL172                             5′CCTCATCCTGATAAACT     5′ TGGTTTTGTGGGCAACATGCTGGTCATCCTCATCCTGATAAACTGCAAAAGGCTGAAGAGCATGACTGACATC        ACCAAAACACCCGTTGTACGACCAGTAGGAGTAGGACTATTTGACGTTTTCCGACTTCTCGTACTGACTGTAG5′ R175                                                  TTTTCCGACTTCTCG5′ R177                                                    TTCCGACTTCTCG5′R178                                                     TCCGACTTCTCGTAC5′R185                                                              TCTCGTACTGACTG5′

As shown below in Table 24, of the 24 pairs tested, 16 yieldeddetectable modification at levels of up to 21%.

TABLE 24 Activity of pairwise combinations of TALEN pairs targeted tolocus 162 of CCR5 % gene modification: +28/+28 pairs +63/+63 pairs R175R177 R178 R185 R175 R177 R178 R185 L161 2% <1 <1 3% L161 4% 18% 12% 8%L164 <1 3% 7% 2% L164 <1 <1 16% 6% L167 <1 1% 2% L167 <1 <1 6% L172 21% L172 5%

Next, the two most active pairs (L172+28/R185+28 and L161+63/R177±63)were introduced into K562 cells with a donor DNA fragment designed totransfer 46 bp insertion bearing a BglII restriction site into thetargeted locus. The donor sequence used is shown in Example 23.

Following insertion, the incorporated tag donor sequence was5′-5′TCATCTTTGGTTTTGTGGGCAACATGCTGGTCATCCTCATCTAGATCAGTGAGTATGCCCTGATGGCGTCTGGACTGGATGCCTCGTCTAGAAAACTGCAAAAGGCTGAAGAGCATGACTGACATCTACCTGCTCAAC-3′ (SEQ ID NO:177) with the unique BglIrestriction site being underlined.

If donor insertion occurred via HDR, the region containing the insertsite can be PCR amplified and then subject to BglI digestion, as isshown below where the top strand shows the sequence of the target site(SEQ ID NO:133) and the bottom strand (SEQ ID NO:134) shows the sequenceof a target had the tag donor inserted. The underlined sequence in thetop strand shows the TALEN binding site while the underlined sequence inthe bottom strand shows the BglI restriction site (SEQ ID NOs: 445through 450):

5′-TCATCTTTGGTTTTGTGGGCAACATGCTGGTCATCCTCATC------------------CTGAT----------------------------AAACTGCAAAAGGCTGAAGAGCATGACTGACATCTACCTGCTCAAC-3′5′-TCATCTTTGGTTTTGTGGGCAACATGCTGGTCATCCTCATC------------------CTGAT----------------------------AAACTGCAAAAGGCTGAAGAGCATGACTGACATCTACCTGCTCAAC-3′5′-TCATCTTTGGTTTTGTGGGCAACATGCTGGTCATCCTCATCTAGATCAGTGAGTAT GCCCTGATGGCGTCTGGACTGGATGCCTCGTCTAGAAAACTGCAAAAGGCTGAAGAGCATGACTGACATCTACCTGCTCAAC-3′

As shown in FIG. 24, PCR products of clones containing an insert had twofragments following BglI digestion. The PCR and BglI digestion scheme isshown in FIG. 24A, while the results are shown in FIG. 24B, and revealedhighly efficient editing. Thus, our TALEN architecture induced efficientgene modification via HDR at an endogenous locus.

Example 13 Examination of Gap Spacing Preferences for Selected TALENArchitectures

To examine the gap spacing preferences of two preferred TALENarchitectures (C+28 C-cap or C+63 C-cap pairs), all TALEN pairscontaining a pairing of C+28/C+28 or C+63/C+63 were sorted for activityaccording to gap spacing. The results are shown in FIG. 25, anddemonstrate that the smaller TALEN proteins, the C+28/C+28 pair, have amore constrained gap spacing preference and are most active on targetswherein the target sequence are separated by gaps of 12 or 13 basepairs. Conversely, the larger TALEN proteins, the C+63/C+63 pairs, shownin FIG. 25B, are active on targets containing gap spacings ranging from12-23 base pairs.

Example 14 Systematic Mapping of Compositions that May be Used to Link aNuclease Domain to a TALE Repeat Array that Provide Highly ActiveNuclease Function

Systematic mapping of compositions that may be used to link a nucleasedomain to a TALE repeat array that provide highly active nucleasefunction. Initially, one TALEN pair was chosen against a single targetwith a defined gap spacing between the two binding domains. The TALENpair chosen was that described in Example 12 as the L538/R557 pair whichwere specific for the CCR5 gene and had an 18 base pair gap spacing. Thedeletions were made as described above such that a truncation seriesresulted in C-caps from C-2 to C+278.

(SEQ ID NO: 178)                 C−2   C+5  C+11  C+17C+22  C+28       C+39            C+55    C+63LTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVADH                 C−1          C+79            C+95                 C+117AQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQLFRRVGVTELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQAC+153                       C+183                         C+213             C+231SLHAFADSLERDLDAPSPTHEGDQRRASSRKRSRSDRAVTGPSAQQSFEVRAPEQRDALHLPLSWRVKRPRTSIGGGLPDPGTPT                                      C+278AADLAASSTVMREQDEDPFAGAADDFPAFNEEELAWLMELLPQ

These truncations were then used to analyze nuclease activity in K562cells using the Cel-I mismatch assay. The results (% NHEJ) are shownbelow in Table 25 and FIG. 26.

TABLE 25 Nuclease activity for fine mapping C-terminal truncations C-capActivity 30° Activity 37° C − 2 18.2% 4.6% C − 1 14.3% 3.3% C + 5 2.1%2.7% C + 11 5.8% 3.2% C + 17 9.2% 5.9% C + 22 5.7% 2.9% C + 28 10.4%3.0% C + 63 48.8% 24.0% C + 79 20.7% 5.0% C + 95 9.8% 2.4% C + 123 14.0%4.2% C + 153 8.1% 0.7% C + 183 7.0% 0.8% C + 213 3.1% 1.7% C + 231 2.2%0.8% C + 278 8.4% 0.7%

The data demonstrate that the peak activity for this nuclease pairagainst this endogenous target occurs when the C-cap is approximatelyC+63, in other words, when the peptideLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVA (SEQ ID NO:451) is used to link the array offull-length TALE repeats to the FokI cleavage domain. In thisexperiment, the nucleases were tested in K652 cells as before and thecells were incubated either at 30° C. or 37° C. The rough estimate ofthe activity ratio of the C+63 C-cap compared to the C+278 was greaterthan 20 times in the 37° C. degree incubation and greater than 6 timesfor the 30° C. incubation.

To more finely characterize those compositions that may be used to linka nuclease domain to an array of full-length TALE repeats that enablehighly active nuclease function at an endogenous locus, additionaltruncations were constructed. A fine series of truncations was assembledcomprising 30 C-caps: C−41, C−35, C−28, C−21, C−16, C−8, C−2, C−1, C+5,C+11, C+17, C+22, C+28, C+34, C+39, C+47, C+55, C+63, C+72, C+79, C+87,C+95, C+109, C+123, C+138, C+153, C+183, C+213, C+231, and C+278. Notethat our C-cap notation starts at residue −20. Thus C−41, C−35, C−28,and C−21 indicates a construct completely lacking a C-cap and with 20,14, 7, or 0 residues removed from the C-terminus of the last full34-residue TALE repeat. Pairs of the constructs were tested against theappropriate target sites with the following gap spacings between targetsites: 0, 2, 4, 7, 10, 14, 18, 23, 28, and 34 base pairs. The pairs weretested against a reporter gene in an SSA assay as well as in a mammaliancell against the endogenous locus. The C-caps are illustrated belowwhere the illustration starts at the last full repeat of a TALE DNAbinding domain and shows the points towards the C terminus.

C-Caps

(SEQ ID NO: 178)           C−41  C−35   C−28   C−21 C−16     C−8   C−2LTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPALE C−1|←          full repeat         →||←    half repeat →|   C+5  C+11  C+17C+22  C+28  C+34 C+39    C+47    C+55    C+63     C+72SIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCC+79  C+87    C+95         C+109         C+123          C+138          C+153HSHPAQAFDDAMTQFGMSRHGLLQLFRRVGVTELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHA                        C+183                         C+213             C+231FADSLERDLDAPSPTHEGDQRRASSRKRSRSDRAVTGPSAQQSFEVRAPEQRDALHLPLSWRVKRPRTSIGGGLPDPGTPTAADLAASSTVMREQDEDPFAGAADDFPAFNEEELAWLMELLPQ. C+278

The target sites for the experiment are shown below, illustrating thepair with a 7 bp gap spacing. Note that the -C−16, C−21, C−28, C−35, andC−41 C-cap constructs remove the RVD in the half repeat for each TALENin the pair and such constructs effectively have a 9 bp gap spacing forthe same target DNA sequence. Target sites for all the other gapspacings tested were constructed by either removing base pairs betweenthe targets or by inserting additional base pairs, depending on the gapspacing to be tested:

Left TALEN binding site-gap--Right TALEN binding siteL538        TCATTACACCTGCAGCT L543             ACACCTGCAGCTCTCATAAAAAGAAGGTCTTCATTACACCTGCAGCTCTCATTTTCCATACAGTCAGTATCAATTCTGGAAGAATTTCCAGACATTTTTCTTCCAGAAGTAATGTGGACGTCGAGAGTAAAAGGTATGTCAGTCATAGTTAAGACCTTCTTAAAGGTCTGT                                          TGTCAGTCATAGT                  R551                                                TCATAGTTAAGACCTTC         R557

The genes encoding the TALEN proteins were assembled as described inExamples 11 and 12 and evaluated by Cel-1 assays. The data are presentedbelow in Table 26A. As shown, the TALE-proteins as described herein cantolerate C-terminal truncations relative to full-length TALE-proteins,including truncations extending into a half repeat and TALE repeatdomain itself without complete loss of functionality against anendogenous locus.

TABLE 26A Effect of C-cap on TALEN activity in mammalian cells L543-R551L538-R551 L543-R557 L538-R557 (7 bp gap) (12 bp gap) (13 bp gap) (18 bpgap) Cel1 Cel1 Cel1 Cel1 C-Cap 37 C. 30 C. DLSSA 37 C. 30 C. DLSSA 37 C.30 C. DLSSA 37 C. 30 C. DLSSA C − 41 0 14 0.60 0 0 0.12 0 0 0.15 0 140.90 C − 35 1 41 0.79 0 0 0.24 0 2 0.28 3 47 1.24 C − 28 0 4 0.23 0 00.11 0 2 0.26 4 65 1.47 C − 21 0 0 0.10 0 0 0.19 0 1 0.21 3 46 1.13 C −16 0 0 0.06 0 0 0.25 0 2 0.27 4 37 1.29 C − 8 0 0 0.02 0 0 0.11 0 0 0.140 8 0.99 C − 2 0 0 0.03 1 29 0.74 2 15 1.14 20 47 1.01 C − 1 0 0 0.13 1854 0.82 1 21 1.29 10 46 1.10 C + 5 0 0 0.08 42 75 0.92 1 13 1.00 0 NA0.12 C + 11 0 0 0.05 69 68 1.02 34 66 1.49 5 5 0.35 C + 17 0 0 0.05 7381 1.03 36 59 1.33 5 13 0.88 C + 22 0 0 0.05 36 74 1.08 11 46 1.09 2 110.93 C + 28 0 0 0.01 21 67 0.65 9 46 1.38 1 10 0.57 C + 34 0 0 0.06 4071 1.15 18 61 1.14 3 18 1.45 C + 39 0 0 0.00 15 32 0.34 4 14 0.79 21 550.85 C + 47 0 0 0.02 0 3 1.31 0 4 1.23 8 41 1.55 C + 55 0 0 0.05 31 710.19 23 69 1.64 7 40 1.17 C + 63 0 0 0.07 4 14 0.83 11 57 1.10 22 640.62 C + 72 0 0 0.03 11 18 0.21 28 61 1.50 15 54 0.82 C + 79 0 0 0.03 15 0.19 4 42 1.24 7 43 0.86 C + 87 0 0 0.04 0 0 0.12 1 12 0.91 4 28 0.78C + 95 0 0 0.04 0 0 0.12 1 8 0.69 0 NA 0.92 C + 109 0 0 0.04 0 0 0.12 01 0.29 1 13 0.83 C + 123 0 0 0.04 0 0 0.13 0 3 0.37 1 24 0.97 C + 138 00 0.04 0 0 0.09 0 9 0.73 3 35 0.56 C + 153 0 0 0.06 0 0 0.07 0 0 0.58 05 0.38 C + 183 0 0 0.07 0 0 0.07 0 0 0.26 0 2 0.35 C + 213 0 0 0.02 0 00.05 0 0 0.18 0 0 0.15 C + 231 0 0 0.04 0 0 0.03 0 0 0.14 0 1 0.08 C +278 0 0 0.05 0 0 0.03 0 0 0.12 0 0 0.72 Note: numbers are the percentNHEJ activity as measured by the Cel-I assay.

In addition, the C-terminal truncations were tested against a reportergene in the DLSSA assay as described below in Example 19. In theseexperiments, four pairs of CCR5-specific TALENs were used in thereporter system where the target site of these pairs was built into theDLSSA reporter plasmids. The binding sites of the four TALENs are shownabove and the TALENs were used as four pairs, L543+R551 (Pair 1),L538+R551 (Pair 2), L543+R557 (Pair 3) L538+R557 (Pair 4). Gap spacingswere varied by insertion or deletion of nucleotides between the bindingsites for the pairs. The data are presented below in Table 26 B-E wherenumeric value indicates the relative fluorescence detected by the DLSSAassay and thus the degree of cleavage. All samples were normalized to acontrol TALEN pair whose binding site is also present on the DLSSAinsert (positive control). Negative control is the assay performed inthe absence of TALENs. The reporter #4 has the exact DNA bindingsequence and the same gap sequences as the endogenous sequence, and thuscan be compared with the Cel-I data at the endogenous locus. The DLSSAdata of the four TALEN pairs from reporter #4 is shown in Table 26A.These data illustrate a general correlation between the results foundwith a reporter system and those observed on an endogenous target areclose and thus the reporter system is useful as a screening tool forcandidate nucleases to test in any endogenous assay. This is a usefultool when working in systems with precious model cells or when theintended target cell type is either not available or difficult to beused for screening purpose. This is also useful tool to develop and tooptimize TALEN technology platform when the target sequences are notavailable in endogenous genome. Active nucleases can be identified byDLSSA and then ported into the endogenous system for final evaluation.

TABLE 26B DLSSA assay with L543-R551 TALEN pair Reporter R1 R2 R3 R4 R5R6 R7 R8 R9 R10 Gap (bp) C-cap 0 2 4 7 10 14 18 23 28 34 C − 41 0.030.05 0.05 0.60 0.23 0.07 0.04 0.05 0.07 0.03 C − 35 0.02 0.06 0.10 0.790.93 0.07 0.33 0.04 0.06 0.00 C − 28 0.02 0.05 0.03 0.23 0.11 0.05 0.010.02 0.04 0.02 C − 21 0.02 0.04 0.01 0.10 0.44 0.06 0.17 0.04 0.04 0.03C − 16 0.01 0.05 0.03 0.06 0.37 0.05 0.15 0.02 0.05 0.02 C − 8 0.03 0.050.04 0.02 0.19 0.47 0.00 0.01 0.04 0.02 C − 2 0.01 0.03 0.00 0.03 1.100.17 0.46 0.05 0.15 0.03 C − 1 0.04 0.04 0.05 0.13 1.23 0.27 0.84 0.140.16 0.04 C + 5 0.04 0.07 0.06 0.08 1.26 0.08 0.03 0.12 0.10 0.08 C + 110.04 0.05 0.03 0.05 1.35 1.00 0.03 0.91 0.09 0.14 C + 17 0.06 0.07 0.040.05 1.39 1.36 0.14 1.30 0.09 0.16 C + 22 0.06 0.03 0.04 0.05 1.06 1.090.23 1.07 0.12 0.15 C + 28 0.01 0.03 0.03 0.01 0.71 0.22 0.16 0.43 0.180.04 C + 34 0.05 0.05 0.04 0.06 0.64 1.33 0.27 1.13 0.21 0.24 C + 390.00 0.02 0.02 0.00 0.06 0.32 0.77 1.02 0.53 0.04 C + 47 0.04 0.04 0.030.02 0.21 1.29 0.43 1.46 0.06 0.15 C + 55 0.04 0.04 0.03 0.05 0.61 1.090.44 1.29 0.25 0.13 C + 63 −0.01 −0.01 0.01 0.07 0.15 0.75 0.83 0.870.69 0.15 C + 72 0.00 0.01 0.01 0.03 0.06 0.88 0.78 1.06 0.55 0.26 C +79 0.02 0.02 0.02 0.03 0.13 0.96 0.93 1.18 0.75 0.27 C + 87 0.03 0.030.02 0.04 0.11 0.87 0.73 0.87 0.43 0.21 C + 95 0.05 0.04 0.03 0.04 0.100.89 0.83 0.94 0.47 0.27 C + 109 0.05 0.03 0.03 0.04 0.09 0.48 0.62 0.470.39 0.30 C + 123 0.04 0.04 0.03 0.04 0.06 0.68 0.65 0.49 0.46 0.26 C +138 0.02 0.03 0.02 0.04 0.08 0.62 1.13 0.95 1.38 0.56 C + 153 0.04 0.040.03 0.06 0.10 0.54 0.86 0.81 1.09 0.40 C + 183 0.05 0.03 0.01 0.07 0.150.24 0.96 0.51 0.90 0.43 C + 213 0.02 0.02 0.01 0.02 0.06 0.15 0.34 0.240.34 0.18 C + 231 0.04 0.03 0.02 0.04 0.05 0.10 0.27 0.21 0.19 0.12 C +278 0.07 0.05 0.03 0.05 0.13 0.12 0.67 0.17 0.69 0.07

TABLE 26C DLSSA assay with L538-R551 TALEN pair Reporter R1 R2 R3 R4 R5R6 R7 R8 R9 R10 Gap (bp) C-cap 5 7 9 12 15 19 23 28 33 39 C − 41 0.220.80 0.60 0.12 0.15 0.11 0.06 0.07 0.07 0.04 C − 35 0.26 0.99 0.85 0.240.30 0.27 0.13 0.08 0.04 0.07 C − 28 0.60 0.60 0.13 0.11 0.08 0.05 0.060.03 0.04 0.03 C − 21 0.10 0.63 0.71 0.19 0.26 0.23 0.08 0.05 0.04 0.06C − 16 0.08 0.55 0.83 0.25 0.35 0.28 0.09 0.06 0.03 0.06 C − 8 0.04 0.060.17 0.11 0.07 0.05 0.02 0.01 0.02 0.06 C − 2 0.35 0.19 0.80 0.74 0.350.71 0.18 0.13 0.10 0.01 C − 1 0.42 0.38 0.80 0.82 0.32 0.88 0.46 0.200.25 0.05 C + 5 0.27 0.18 0.15 0.92 0.09 0.13 0.37 0.07 0.19 0.02 C + 110.28 0.07 0.18 1.02 0.35 0.27 1.14 0.11 0.31 0.04 C + 17 0.21 0.09 0.261.03 0.79 0.38 1.15 0.13 0.43 0.06 C + 22 0.14 0.06 0.13 1.08 0.87 0.341.07 0.14 0.39 0.07 C + 28 0.20 0.04 0.12 0.65 0.51 0.29 0.64 0.08 0.160.00 C + 34 0.08 0.06 0.14 1.15 0.96 0.75 1.08 0.28 0.53 0.11 C + 390.11 −0.02 0.02 0.34 0.65 0.40 1.03 0.72 0.33 0.05 C + 47 0.11 0.13 0.301.31 1.17 0.75 1.33 0.31 0.51 0.13 C + 55 0.23 0.08 0.14 0.19 0.53 0.770.91 0.76 0.54 0.09 C + 63 0.33 0.07 0.18 0.83 0.71 0.90 1.19 0.26 0.370.02 C + 72 0.26 −0.01 0.03 0.21 0.50 0.55 0.78 0.64 0.32 0.17 C + 790.25 0.01 0.04 0.19 0.59 0.53 0.95 0.68 0.64 0.24 C + 87 0.15 0.00 0.040.12 0.64 0.36 0.75 0.51 0.46 0.23 C + 95 0.10 0.01 0.06 0.12 0.74 0.400.71 0.46 0.56 0.24 C + 109 0.08 0.03 0.05 0.12 0.67 0.31 0.50 0.34 0.440.33 C + 123 0.06 0.06 0.07 0.13 0.84 0.45 0.61 0.44 0.46 0.33 C + 1380.48 0.01 0.03 0.09 0.67 1.02 1.15 1.13 0.96 0.30 C + 153 0.35 0.01 0.030.07 0.63 0.78 1.09 0.91 0.83 0.30 C + 183 0.45 0.05 0.06 0.07 0.64 0.621.01 0.99 0.95 0.38 C + 213 0.24 0.02 0.02 0.05 0.58 0.35 0.66 0.62 0.620.28 C + 231 0.12 0.01 0.02 0.03 0.53 0.12 0.29 0.25 0.32 0.12 C + 2780.07 0.03 0.03 0.03 0.49 0.19 0.17 0.51 0.15 0.07

TABLE 26D DLSSA assay with L543-R557 TALEN pair Reporter R1 R2 R3 R4 R5R6 R7 R8 R9 R10 Gap (bp) C-cap 6 8 10 13 16 20 24 29 34 40 C − 41 0.620.85 0.32 0.15 0.26 0.08 −0.02 0.01 0.03 0.15 C − 35 0.64 0.99 0.93 0.280.71 0.24 0.03 0.07 0.03 0.55 C − 28 0.21 0.77 1.27 0.26 0.34 0.38 −0.010.04 0.04 0.31 C − 21 0.07 0.59 0.76 0.21 0.31 0.26 0.02 0.07 0.07 0.21C − 16 0.07 1.11 0.83 0.27 0.38 0.32 0.07 0.11 0.13 0.15 C − 8 0.10 1.511.29 0.14 0.16 0.30 0.00 0.08 0.13 0.09 C − 2 0.36 1.62 1.72 1.14 1.710.80 0.13 0.14 0.10 0.25 C−1 0.33 1.65 1.43 1.29 1.39 1.00 0.15 0.160.10 0.23 C + 5 0.15 0.11 1.11 1.00 0.36 0.64 0.03 0.00 0.19 0.15 C + 110.11 0.10 1.02 1.49 0.75 0.75 0.29 0.04 0.31 0.10 C + 17 0.10 0.00 1.051.33 0.84 0.86 0.59 0.08 0.40 0.08 C + 22 0.08 −0.01 0.82 1.09 0.98 0.560.42 0.10 0.28 0.08 C + 28 0.14 0.08 1.14 1.38 1.65 0.64 0.37 0.18 0.230.22 C + 34 0.06 0.04 0.78 1.14 1.09 0.78 0.55 0.18 0.53 0.10 C + 390.16 0.04 0.06 0.79 1.86 0.79 0.30 0.40 0.05 0.25 C + 47 0.09 0.08 0.471.23 1.30 0.84 0.73 0.31 0.55 0.14 C + 55 0.26 0.10 0.48 1.64 2.50 1.111.03 0.25 0.65 0.27 C + 63 0.19 0.04 0.14 1.10 2.47 0.85 0.87 0.67 0.440.69 C + 72 0.21 0.34 0.22 1.50 2.19 1.00 0.84 0.49 0.51 0.77 C + 790.19 0.05 0.11 1.24 1.49 0.71 0.53 0.28 0.35 0.61 C + 87 0.11 0.03 0.080.91 1.25 0.46 0.33 0.21 0.27 0.32 C + 95 0.08 0.02 0.07 0.69 0.99 0.510.43 0.29 0.35 0.30 C + 109 0.08 0.11 0.14 0.29 0.85 0.31 0.27 0.31 0.390.30 C + 123 0.08 0.08 0.08 0.37 0.94 0.36 0.37 0.42 0.51 0.28 C + 1380.29 0.17 0.19 0.73 3.13 0.56 1.19 0.53 0.63 1.17 C + 153 0.24 0.16 0.110.58 2.16 0.57 1.09 0.46 0.52 0.98 C + 183 0.28 0.19 0.15 0.26 2.32 0.380.78 0.26 0.44 0.88 C + 213 0.22 0.10 0.05 0.18 1.32 0.20 0.32 0.10 0.240.40 C + 231 0.13 0.11 0.04 0.14 0.92 0.09 0.17 0.04 0.11 0.12 C + 2780.08 0.11 0.04 0.12 0.76 0.37 0.18 0.42 0.10 0.14

TABLE 26E DLSSA assay with L538-R557 TALEN pair Reporter R1 R2 R3 R4 R5R6 R7 R8 R9 R10 Gap (bp) C-cap 11 13 15 18 21 25 29 34 39 45 C − 41 0.450.28 1.26 0.90 0.07 0.34 0.17 0.02 0.17 0.09 C − 35 0.94 0.34 1.82 1.240.27 0.52 0.32 0.05 0.26 0.16 C − 28 1.21 0.71 2.99 1.47 0.38 0.11 0.370.02 0.09 0.10 C − 21 1.03 0.03 1.03 1.13 0.01 0.03 0.39 0.03 0.16 0.08C − 16 0.77 0.71 1.30 1.29 0.43 0.16 0.48 0.07 0.14 0.15 C − 8 1.01 1.000.61 0.99 0.46 0.02 0.20 0.02 0.04 0.05 C − 2 0.94 0.78 1.43 1.01 0.750.39 0.29 0.06 0.17 0.06 C − 1 1.20 0.88 1.81 1.10 1.04 0.76 0.41 0.240.18 0.22 C + 5 1.29 0.75 0.38 0.12 0.65 0.11 0.02 0.40 0.06 0.17 C + 111.39 1.00 0.97 0.35 0.90 0.46 0.08 0.53 0.08 0.24 C + 17 1.34 0.85 1.950.88 1.04 0.94 0.20 0.61 0.06 0.29 C + 22 1.58 1.32 1.70 0.93 1.03 0.850.23 0.42 0.07 0.22 C + 28 0.78 0.63 1.44 0.57 0.52 0.61 0.08 0.15 0.150.09 C + 34 1.35 1.58 2.05 1.45 1.27 0.92 0.48 0.47 0.16 0.20 C + 390.01 0.49 1.49 0.85 0.61 0.25 0.47 0.03 0.12 0.05 C + 47 1.24 1.10 1.711.55 1.45 1.07 0.54 0.52 0.11 0.31 C + 55 1.14 1.48 1.96 1.17 1.05 1.420.36 0.55 0.36 0.42 C + 63 0.03 0.42 1.11 0.62 0.67 0.76 0.41 0.14 0.350.07 C + 72 0.09 0.79 1.43 0.82 0.75 1.23 0.52 0.27 0.43 0.17 C + 790.07 0.90 1.26 0.86 0.90 1.18 0.50 0.19 0.38 0.20 C + 87 0.06 0.89 1.200.78 0.89 0.92 0.40 0.22 0.25 0.15 C + 95 0.05 0.91 2.72 0.92 0.93 0.770.41 0.23 0.22 0.19 C + 109 0.08 0.57 0.90 0.83 0.90 0.66 0.62 0.37 0.330.18 C + 123 0.05 0.93 0.88 0.97 0.99 0.58 0.57 0.35 0.20 0.22 C + 1380.05 0.42 1.19 0.56 0.57 1.03 0.22 0.26 0.77 0.23 C + 153 0.04 0.63 0.780.38 0.61 0.84 0.19 0.23 0.57 0.25 C + 183 0.04 0.15 0.68 0.35 0.39 0.780.14 0.25 0.60 0.29 C + 213 0.03 0.13 0.37 0.15 0.29 0.42 0.11 0.15 0.320.24 C + 231 0.00 0.14 0.29 0.08 0.19 0.24 0.03 0.06 0.10 0.10 C + 2780.03 0.18 0.55 0.72 0.71 0.37 0.90 0.08 0.21 0.08

Thus, the Cel-I and DLSSA results indicate that these proteins havesubstantial and robust activity when the appropriate C-cap is used andan N-cap is present. Further, gap spacings may play a role in themaximum activity observed with smaller gap spacings being active with asmaller subset of C-terminal truncations as compared to larger gapspacings. We also note that the relative DLSSA activity does not appearto be linearly related to the endogenous activity for the same TALENsobtained at the same temperature (37 degrees Celsius). The reporterresults yield a significantly higher relative activity for constructswith C+153, C+183, C+213, C+231, and C+278 C-caps than observed at thenative endogenous locus of human cells. Thus activity in reportersystems, even reporter systems in mammalian cells, does not necessarilypredict the activity at the native endogenous in mammalian cells.

Example 15 Novel (Atypical) RVDs

Alternative (atypical) RVDs were explored to determine if other aminoacids at the positions that determine DNA binding specificity could bealtered. A TALE binding domain was constructed whose binding activitywas shown by SELEX and ELISA to be sensitive to a mismatch at the middleposition. This protein bound the sequence 5′-TTGACAATCCT-3′(SEQ IDNO:178) and displayed little binding activity against the sequences5′-TTGACCATCCT-3′ (SEQ ID NO:179), 5′-TTGACGATCCT-3′ (SEQ ID NO:180), or5′-TTGACTATCCT-3′ (SEQ ID NO:181) (ELISA data shown in FIG. 27). Thesetargets are referred to as the CXA targets denoting the middle tripletnucleic acid, where X is either A, C, T or G.

This TALE backbone was then used to characterize the DNA-bindingspecificity of alternative RVDs (amino acids 12 and 13) for the TALErepeat that targets the base at the 6^(th) position. The two codons thatencode this RVD were randomized and clones were screened by sequencingto ensure that the complete repeat units were present. Correct cloneswere then analyzed by a DNA-binding ELISA against four versions of thetarget sequence wherein each sequence had either an A, C, T or G at theposition the novel (i.e., atypical) RVD would interact with (i.e.TTGACCAATCCT (SEQ ID NO:178), TTGACCATCCT (SEQ ID NO:182), TTGACTATCCT(SEQ ID NO:183) or TTGACGATCCT (SEQ ID NO:184)). Results from thesestudies are shown below in Table 27A and demonstrate that this assayidentified that the RVD VG can specifically interact with T, RG caninteract with T, TA can interact with T and AA can interact with A, Cand T.

TABLE 27A Exemplary novel RVDs Target (ELISA units) RVD Note CAA CCA CGACTA AE 9 10 9 11 GR 10 9 14 29 TR 17 47 12 308 PR 8 7 8 13 LH 9 8 9 12VG 34 16 14 596 RE 23 24 9 24 RG 487 314 169 1240 RC 12 9 7 8 TA 89 12516 755 AA 433 447 84 750 QR 11 8 10 13 LR 11 9 8 12 SR 13 11 23 27 GE 99 ND 13 VR 33 14 ND 26 NI CAA Binder 1105 45 15 13 Control NN CGA Binder1305 10 1730 13 Control negative 7 Control

Following these initial studies, an analysis was done with all potentialRVD combinations and several were identified with high activity andspecificity. In addition, RVDs were identified that bound equally wellto all bases tested. The data are presented in numeric format below inTable 27B and also in FIG. 28. In the data shown below, all data wasbackground corrected by subtracting the background ELISA signal and thennormalized to the average value of NI with the CAA site, HD with the CCAsite, NN with the CGA site, and NG with the CTA site.

TABLE 27B Novel RVDs RVD CAA CCA CGA CTA RVD CAA CCA CGA CTA AA 0.340.35 0.06 0.61 AE 0.00 0.00 0.00 0.00 CA 0.18 0.22 0.02 0.89 CE 0.010.05 0.00 0.04 DA 0.12 0.22 0.02 0.55 DE 0.01 0.01 0.00 0.01 EA 0.260.58 0.08 1.20 EE 0.01 0.04 0.00 0.03 FA 0.22 0.26 0.03 1.25 FE 0.010.02 0.00 0.05 GA 0.07 0.04 0.01 0.53 GE 0.00 0.00 0.00 0.00 HA 0.460.53 0.24 1.54 HE 0.03 0.04 0.00 0.08 IA 0.15 0.24 0.02 1.20 IE 0.000.03 0.00 0.07 KA 0.85 0.98 0.25 1.63 KE 0.00 0.01 0.00 0.01 LA 0.020.02 0.00 0.51 LE 0.00 0.01 0.00 0.03 MA 0.12 0.11 0.02 0.66 ME 0.000.01 0.00 0.03 NA 0.59 0.67 0.41 1.72 NE 0.02 0.02 0.00 0.02 PA 0.000.00 0.00 0.04 PE 0.00 0.00 0.00 0.03 QA 0.20 0.19 0.03 1.24 QE 0.010.03 0.00 0.04 RA 0.73 0.89 0.53 1.64 RE 0.01 0.01 0.00 0.01 SA 0.420.44 0.06 1.05 SE 0.00 0.01 0.00 0.01 TA 0.15 0.20 0.01 0.76 TE 0.020.08 0.00 0.09 VA 0.21 0.25 0.04 1.06 VE 0.01 0.05 0.00 0.08 WA 0.490.35 0.05 1.40 WE 0.00 0.01 0.00 0.03 YA 0.29 0.23 0.04 1.36 YE 0.010.02 0.00 0.04 AC 0.34 0.29 0.06 0.11 AF 0.02 0.00 0.00 0.02 CC 0.200.32 0.08 0.19 CF 0.01 0.00 0.00 0.02 DC 0.11 0.11 0.02 0.03 DF 0.000.00 0.00 0.02 EC 0.14 0.19 0.04 0.07 EF 0.01 0.00 0.00 0.03 FC 0.080.14 0.03 0.05 FF 0.00 0.00 0.00 0.03 GC 0.07 0.05 0.01 0.05 GF 0.020.00 0.00 0.03 HC 0.74 0.85 0.54 0.33 HF 0.04 0.00 0.00 0.00 IC 0.070.20 0.02 0.15 IF 0.00 0.00 0.00 0.02 KC 0.68 0.82 0.40 0.51 KF 0.000.00 0.00 0.00 LC 0.04 0.04 0.01 0.02 LF 0.00 0.00 0.00 0.02 MC 0.050.06 0.01 0.04 MF 0.00 0.00 0.00 0.01 NC 0.45 0.51 0.09 0.05 NF 0.010.00 0.00 0.00 PC 0.01 0.02 0.00 0.01 PF 0.00 0.00 0.00 0.02 QC 0.140.17 0.03 0.09 QF 0.01 0.00 0.00 0.02 RC 0.00 0.00 0.00 0.00 RF 0.090.00 0.00 0.02 SC 0.35 0.29 0.09 0.17 SF 0.01 0.01 0.00 0.05 TC 0.090.12 0.03 0.17 TF 0.01 0.00 0.00 0.02 VC 0.10 0.26 0.04 0.18 VF 0.000.00 0.00 0.02 WC 0.07 0.08 0.02 0.03 WF 0.00 0.00 0.00 0.02 YC 0.120.14 0.05 0.05 YF 0.00 0.00 0.00 0.02 AD 0.02 0.40 0.00 0.01 AG 0.190.12 0.07 0.80 CD 0.00 0.24 0.00 0.01 CG 0.16 0.07 0.03 0.73 DD 0.000.13 0.00 0.00 DG 0.02 0.01 0.00 0.13 ED 0.01 0.36 0.00 0.02 EG 0.060.03 0.01 0.39 FD 0.00 0.07 0.00 0.00 FG 0.04 0.01 0.00 0.37 GD 0.000.04 0.02 0.01 GG 0.49 0.35 0.25 1.52 HD 0.15 1.00 0.00 0.09 HG 0.380.11 0.11 1.49 ID 0.00 0.08 0.00 0.01 IG 0.04 0.02 0.01 0.68 KD 0.060.61 0.00 0.05 KG 0.47 0.27 0.20 1.29 LD 0.00 0.01 0.00 0.01 LG 0.020.02 0.00 0.31 MD 0.00 0.04 0.00 0.00 MG 0.20 0.14 0.05 0.95 ND 0.060.73 0.00 0.02 NG 0.48 0.14 0.13 1.30 PD 0.00 0.00 0.01 0.00 PG 0.010.01 0.01 0.11 QD 0.01 0.24 0.00 0.00 QG 0.21 0.11 0.05 1.09 RD 0.220.79 0.00 0.01 RG 0.32 0.26 0.12 0.87 SD 0.01 0.28 0.00 0.01 SG 0.360.23 0.20 1.23 TD 0.01 0.10 0.00 0.06 TG 0.24 0.14 0.06 0.96 VD 0.000.11 0.00 0.02 VG 0.05 0.02 0.01 0.56 WD 0.01 0.10 0.00 0.01 WG 0.050.01 0.01 0.44 YD 0.01 0.28 0.00 0.01 YG 0.12 0.02 0.01 0.79 AP 0.170.28 0.04 0.54 AS 0.67 0.35 0.55 0.36 CP 0.13 0.23 0.04 0.96 CS 0.450.36 0.27 0.60 DP 0.06 0.10 0.01 0.25 DS 0.24 0.17 0.17 0.09 EP 0.100.20 0.01 0.54 ES 0.37 0.35 0.27 0.31 FP 0.07 0.15 0.01 0.66 FS 0.240.18 0.12 0.30 GP 0.04 0.07 0.01 0.11 GS 0.50 0.11 0.27 0.16 HP 0.710.82 0.16 0.93 HS 0.77 0.79 0.69 0.80 IP 0.04 0.13 0.00 0.84 IS 0.180.32 0.08 0.75 KP 0.55 0.77 0.13 1.37 KS 0.95 0.78 0.74 0.81 LP 0.020.08 0.00 0.46 LS 0.29 0.15 0.09 0.27 MP 0.01 0.03 0.00 0.25 MS 0.300.16 0.09 0.29 NP 0.07 0.17 0.06 0.81 NS 0.51 0.26 0.71 0.54 PP 0.000.00 0.00 0.04 PS 0.01 0.00 0.00 0.03 QP 0.09 0.14 0.01 0.56 QS 0.590.41 0.30 0.44 RP 0.77 0.76 0.15 1.40 RS 0.64 0.59 0.63 0.52 SP 0.310.39 0.05 1.19 SS 0.37 0.23 0.38 0.48 TP 0.16 0.20 0.01 1.20 TS 0.170.14 0.12 0.48 VP 0.07 0.13 0.01 0.87 VS 0.29 0.29 0.15 0.67 WP 0.030.06 0.00 0.31 WS 0.36 0.20 0.12 0.26 YP 0.08 0.16 0.01 0.68 YS 0.620.40 0.23 0.52 AQ 0.04 0.03 0.13 0.05 AT 1.29 0.58 0.76 0.56 CQ 0.030.03 0.23 0.08 CT 1.01 0.64 0.36 0.78 DQ 0.01 0.04 0.05 0.01 DT 0.900.27 0.30 0.05 EQ 0.01 0.06 0.10 0.02 ET 1.31 0.60 0.35 0.24 FQ 0.000.01 0.05 0.02 FT 0.87 0.72 0.54 0.27 GQ 0.01 0.02 0.03 0.04 GT 0.780.19 0.50 0.14 HQ 0.22 0.17 0.49 0.12 HT 0.72 0.68 1.24 0.67 IQ 0.010.01 0.09 0.12 IT 0.46 0.34 0.17 0.40 KQ 0.13 0.10 0.40 0.10 KT 1.000.81 0.83 0.67 LQ 0.00 0.00 0.01 0.02 LT 0.43 0.11 0.09 0.05 MQ 0.000.00 0.03 0.03 MT 0.37 0.13 0.11 0.19 NQ 0.03 0.04 0.18 0.02 NT 0.820.41 0.99 0.29 PQ 0.00 0.00 0.00 0.01 PT 0.02 0.01 0.00 0.04 QQ 0.020.03 0.11 0.03 QT 0.64 0.38 0.43 0.42 RQ 0.28 0.09 0.49 0.20 RT 0.620.43 0.51 0.35 SQ 0.04 0.06 0.14 0.10 ST 0.62 0.31 0.41 0.44 TQ 0.020.02 0.14 0.09 TT 0.46 0.23 0.14 0.58 VQ 0.02 0.02 0.11 0.15 VT 0.330.31 0.14 0.55 WQ 0.01 0.01 0.04 0.03 WT 0.33 0.16 0.09 0.09 YQ 0.020.03 0.14 0.05 YT 0.39 0.28 0.15 0.18 AR 0.00 0.00 0.00 0.01 AV 0.210.12 0.10 0.10 CR 0.00 0.00 0.01 0.02 CV 0.27 0.22 0.12 0.16 DR 0.000.00 0.00 0.01 DV 0.15 0.09 0.06 0.01 ER 0.00 0.00 0.01 0.01 EV 0.180.14 0.06 0.02 FR 0.00 0.00 0.00 0.00 FV 0.09 0.07 0.05 0.01 GR 0.000.00 0.01 0.02 GV 0.10 0.08 0.05 0.05 HR 0.00 0.00 0.03 0.02 HV 0.560.49 0.25 0.02 IR 0.00 0.00 0.00 0.03 IV 0.10 0.16 0.04 0.09 KR 0.000.00 0.03 0.04 KV 0.75 0.58 0.28 0.12 LR 0.00 0.00 0.00 0.00 LV 0.060.04 0.02 0.01 MR 0.00 0.00 0.00 0.00 MV 0.08 0.08 0.04 0.02 NR 0.010.00 0.03 0.01 NV 0.37 0.16 0.07 0.01 PR 0.00 0.00 0.00 0.00 PV 0.000.00 0.00 0.01 QR 0.00 0.00 0.00 0.00 QV 0.17 0.14 0.08 0.04 RR 0.010.00 0.08 0.05 RV 0.54 0.43 0.32 0.07 SR 0.00 0.00 0.01 0.02 SV 0.290.17 0.14 0.14 TR 0.00 0.00 0.01 0.05 TV 0.01 0.00 0.00 0.01 VR 0.020.01 0.00 0.01 VV 0.16 0.20 0.07 0.14 WR 0.00 0.00 0.00 0.01 WV 0.100.08 0.02 0.01 YR 0.00 0.00 0.01 0.01 YV 0.15 0.11 0.06 0.02 AH 0.040.02 0.33 0.04 AL 0.04 0.00 0.00 0.05 CH 0.02 0.02 0.04 0.12 CL 0.070.00 0.00 0.03 DH 0.01 0.01 0.36 0.03 DL 0.01 0.00 0.00 0.03 EH 0.020.03 0.17 0.13 EL 0.02 0.00 0.00 0.03 FH 0.00 0.00 0.02 0.04 FL 0.010.00 0.00 0.02 GH 0.00 0.01 0.12 0.01 GL 0.02 0.01 0.00 0.04 HH 0.050.07 0.37 0.17 HL 0.04 0.00 0.00 0.00 IH 0.00 0.01 0.02 0.07 IL 0.020.00 0.00 0.03 KH 0.01 0.01 0.12 0.09 KL 0.07 0.00 0.00 0.01 LH 0.000.00 0.00 0.00 LL 0.00 0.00 0.00 0.02 MH 0.00 0.01 0.01 0.03 ML 0.010.00 0.00 0.01 NH 0.03 0.02 0.18 0.09 NL 0.02 0.00 0.00 0.00 PH 0.000.00 0.00 0.01 PL 0.00 0.00 0.00 0.03 QH 0.02 0.03 0.09 0.09 QL 0.020.00 0.00 0.00 RH 0.05 0.03 0.39 0.05 RL 0.14 0.01 0.00 0.01 SH 0.020.02 0.06 0.06 SL 0.02 0.01 0.01 0.07 TH 0.01 0.02 0.11 0.08 TL 0.060.09 0.08 0.16 VH 0.01 0.01 0.01 0.11 VL 0.01 0.00 0.00 0.00 WH 0.000.00 0.01 0.01 WL 0.02 0.02 0.01 0.12 YH 0.01 0.01 0.02 0.02 YL 0.030.03 0.01 0.08 AI 0.33 0.02 0.00 0.03 AM 0.06 0.00 0.05 0.03 CI 0.460.04 0.01 0.04 CM 0.06 0.00 0.07 0.02 DI 0.18 0.01 0.01 0.00 DM 0.030.00 0.02 0.01 EI 0.37 0.06 0.00 0.01 EM 0.09 0.16 0.04 0.20 FI 0.130.00 0.00 0.00 FM 0.02 0.00 0.03 0.00 GI 0.07 0.01 0.01 0.01 GM 0.040.06 0.04 0.16 HI 0.67 0.10 0.04 0.04 HM 0.30 0.28 0.00 0.03 II 0.050.01 0.00 0.03 IM 0.07 0.13 0.03 0.14 KI 0.75 0.11 0.02 0.04 KM 0.030.03 0.00 0.01 LI 0.01 0.00 0.00 0.00 LM 0.05 0.08 0.04 0.17 MI 0.050.00 0.00 0.01 MM 0.02 0.04 0.03 0.10 NI 0.60 0.04 0.02 0.02 NM 0.050.06 0.00 0.00 PI 0.01 0.00 0.00 0.01 PM 0.01 0.02 0.03 0.13 QI 0.300.05 0.00 0.04 QM 0.11 0.12 0.01 0.22 RI 0.65 0.05 0.02 0.02 RM 0.170.09 0.00 0.02 SI 0.29 0.02 0.00 0.03 SM 0.11 0.16 0.03 0.17 TI 0.320.11 0.00 0.05 TM 0.04 0.08 0.03 0.05 VI 0.15 0.04 0.00 0.07 VM 0.040.09 0.04 0.05 WI 0.06 0.00 0.00 0.01 WM 0.02 0.04 0.02 0.03 YI 0.150.01 0.00 0.01 YM 0.05 0.11 0.05 0.06 AK 0.00 0.00 0.21 0.01 AN 0.510.00 0.87 0.01 CK 0.00 0.00 0.10 0.00 CN 0.17 0.00 0.49 0.02 DK 0.000.00 0.15 0.00 DN 0.12 0.00 0.37 0.01 EK 0.00 0.00 0.11 0.00 EN 0.190.00 0.49 0.01 FK 0.00 0.00 0.04 0.00 FN 0.12 0.00 0.37 0.01 GK 0.010.00 0.09 0.04 GN 0.12 0.00 0.32 0.02 HK 0.00 0.00 0.06 0.00 HN 0.500.00 0.86 0.01 IK 0.00 0.00 0.07 0.01 IN 0.05 0.00 0.17 0.03 KK 0.000.00 0.08 0.01 KN 0.71 0.00 1.00 0.02 LK 0.00 0.00 0.01 0.00 LN 0.030.00 0.15 0.00 MK 0.00 0.00 0.01 0.00 MN 0.08 0.00 0.21 0.01 NK 0.000.00 0.15 0.00 NN 0.47 0.00 0.81 0.00 PK 0.00 0.00 0.00 0.00 PN 0.000.00 0.02 0.01 QK 0.00 0.00 0.15 0.01 QN 0.16 0.00 0.48 0.02 RK 0.000.00 0.12 0.00 RN 0.31 0.00 0.55 0.01 SK 0.00 0.01 0.07 0.02 SN 0.430.01 0.92 0.02 TK 0.00 0.00 0.09 0.01 TN 0.12 0.00 0.32 0.03 VK 0.000.00 0.01 0.00 VN 0.08 0.00 0.30 0.02 WK 0.00 0.00 0.02 0.00 WN 0.130.00 0.36 0.01 YK 0.00 0.00 0.04 0.00 YN 0.18 0.00 0.48 0.01 AW 0.000.00 0.00 0.01 AY 0.02 0.00 0.00 0.01 CW 0.00 0.00 0.00 0.01 CY 0.000.00 0.00 0.01 DW 0.00 0.00 0.00 0.01 DY 0.00 0.00 0.00 0.01 EW 0.000.00 0.00 0.00 EY 0.00 0.00 0.00 0.01 FW 0.00 0.00 0.00 0.01 FY 0.000.00 0.00 0.01 GW 0.00 0.00 0.00 0.02 GY 0.01 0.00 0.01 0.02 HW 0.000.00 0.00 0.00 HY 0.01 0.00 0.00 0.00 IW 0.00 0.01 0.01 0.01 IY 0.000.00 0.00 0.01 KW 0.00 0.00 0.00 0.00 KY 0.03 0.00 0.00 0.00 LW 0.000.00 0.00 0.01 LY 0.00 0.00 0.00 0.01 MW 0.00 0.00 0.00 0.01 MY 0.000.00 0.00 0.00 NW 0.00 0.00 0.00 0.00 NY 0.03 0.00 0.00 0.00 PW 0.000.00 0.00 0.00 PY 0.00 0.00 0.00 0.00 QW 0.01 0.00 0.00 0.00 QY 0.010.00 0.00 0.00 RW 0.00 0.00 0.01 0.01 RY 0.06 0.01 0.00 0.00 SW 0.010.03 0.00 0.18 SY 0.01 0.01 0.00 0.02 TW 0.00 0.00 0.00 0.01 TY 0.010.00 0.00 0.00 VW 0.00 0.00 0.00 0.00 VY 0.00 0.00 0.00 0.00 WW 0.000.00 0.01 0.01 WY 0.00 0.00 0.00 0.00 YW 0.00 0.00 0.00 0.00 YY 0.000.00 0.00 0.00

This data is also presented in FIG. 28 where the data is shown in a20×20 grid. The first amino acid of the RVD (position 12) is indicatedto the left of the grid and the second amino acid of the RVD (position13) is indicated above the grid. The size of the letters A, C, G, and Tin each grid is scaled based on the square root of the normalized ELISAsignal for the CAA site, CCA site, and CGA site and CTA siterespectively. The boxed RVDs indicate frequently occurring natural RVDsfound in TALE proteins encoded by Xanthomonas. Many RVDs have improvedDNA binding properties with respect to the naturally occurring HD, NI,NG, NS, NN, IG, HG, and NK RVDs. Exemplary novel RVDs and their cognatenucleotide bases include where N represents positive interaction withall bases:

A: RI, KI, HI

C: ND, KD, AD

G: DH, SN, AK, AN, DK, HN

T: VG, IA, IP, TP, QA, YG, LA, SG, HA, NA, GG, KG, QG

N: KS, AT, KT, RA.

Studies were also undertaken to purposely alter the RVD sequences tospecific sequences hypothesized to be candidate novel binders through ananalysis of the known RVDs. Thus, the following RVDs have been tested:

RVD Intended target NV, NT, NL, HI, SI, LI A HE, NE, SE, ND, SD, LD CHR, NR, SR, HH, NH, SH, HN, HK, SN, SK, LN, LK G NP, NA, HA, HG, SG, LGT

Oligonucleotides were made to allow the specific alteration of the TALEconstruct described above. These specific oligonucleotides are thencloned into the expression vectors and assembled as described in Example11, and resultant protein extracts are analyzed by DNA-binding ELISA andSELEX to determine the binding characteristics of the RVDs.

Twelve of these TALE DNA binding domains comprising the atypical RVDswere subjected to SELEX analysis as described above. The results fromthe SELEX analysis are shown below in Table 28. In the table, the datafor the natural RVD (in bold in the ‘RVD’ column) is presented alongwith the exemplary novel RVD, and show that in many cases, the novel RVDdemonstrates equal or greater preference for the targeted bases ascompared with the natural RVD.

TABLE 28 SELEX results from novel RVDs:

These RVDs were then tested for activity in the context of a full lengthTALEN. A CCR5-specific 18 repeat TALEN was produced with all novel RVDsfor comparison with the CCR5-specific TALEN described in Example 12. Thetarget sites for this TALEN pair is reshown below. The 101041 TALENmonomer was the partner that was modified while the 101047 partner wasleft with all natural RVDs:

     101041(L538)5′-GTCTTCATTACACCTGCAGCTCTCATTTTCCATACAGTCAGTATCAATTCTGGAAGAATTTCCAGACATT   CAGAAGTAATGTGGACGTCGAGAGTAAAAGGTATGTCAGTCATAGTTAAGACCTTCTTAAAGGTCTGTAA-5′                                            101047 (R557)

In addition, CCR5-specific TALENs comprising both typical and novel(atypical) RVDs were also constructed in CCR5 specific TALENs in whichnovel RVDs were substituted of all one type, for example, all RVDsrecognizing ‘T’ or ‘A’. The code described previously in Examples 11 and12 for the typical RVDs was used, i.e. A=NI, C=HD, G=NN, T=NG. For thenovel RVDs, the following were tested in this initial analysis: A=HI, NIor KI; C=ND, KD, cND; G=SN, AK, DH, cHN, KN; T=TP, IA, VG, SGgs, or IP.When lower case letters are used, these indicate alterations of thepositions adjacent to the RVD positions, for example “cND’ indicatesthat positions 11, 12 and 13 in the repeat unit were altered. For thesestudies, candidate RVDs were chosen by the data presented in Table 27Band used to create proof of principal proteins. Additional TALE proteinsmay be constructed using alternative atypical RVDs from the entire set.In addition, atypical RVDs may be chosen such that a mixture of RVDsspecifying a base may be created (e.g. one TALEN protein may beconstructed using both TP and IA RVDs to specify ‘T’ in differentpositions).

The RVD sequences for the repeat units are shown below in Tables 29A-29Cand all mutated positions are indicated in bold font.

TABLE 29A All novel (atypical) RVD substitution RVD Substitution TALEN TC A T T A C A C C T G C A G C T Full 101726 TP ND HI TP TP HI ND HI NDND TP SN ND HI SN ND TP 101727 IA ND HI IA IA HI ND HI ND ND IA SN ND HISN ND IA 101728 VG ND HI VG VG HI ND HI ND ND VG SN ND HI SN ND VG101729 SGgs ND HI SGgs SGgs HI ND HI ND ND SGgs SN ND HI SN ND SGgs101730 TP ND HI TP TP HI ND HI ND ND TP AK ND HI AK ND TP 101731 IA NDHI IA IA HI ND HI ND ND IA AK ND HI AK ND IA 101732 VG ND HI VG VG HI NDHI ND ND VG AK ND HI AK ND VG 101733 SGgs ND HI SGgs SGgs HI ND HI ND NDSGgs AK ND HI AK ND SGgs 101734 TP ND HI TP TP HI ND HI ND ND TP DH NDHI DH ND TP 101735 IA ND HI IA IA HI ND HI ND ND IA DH ND HI DH ND IA101736 VG ND HI VG VG HI ND HI ND ND VG DH ND HI DH ND VG 101737 SGgs NDHI SGgs SGgs HI ND HI ND ND SGgs DH ND HI DH ND SGgs 101738 TP KD KI TPTP KI KD KI KD KD TP SN KD KI SN KD TP 101739 IA KD KI IA IA KI KD KI KDKD IA SN KD KI SN KD IA 101740 TP KD KI TP TP KI KD KI KD KD TP AK KD KIAK KD TP 101741 IA KD KI IA IA KI KD KI KD KD IA AK KD KI AK KD IA Alltypical 101041 NG HD NI NG NG NI HD NI HD HD NG nNN HD NI nNN HD NG

TABLE 29B Type substitutions Type 101742 NG HD HI NG NG HI HD HI HD HDNG nNN HD HI nNN HD NG 101743 NG HD KI NG NG KI HD KI HD HD NG nNN HD KInNN HD NG 101744 NG HD RI NG NG RI HD RI HD HD NG nNN HD RI nNN HD NG101745 NG ND NI NG NG NI ND NI ND ND NG nNN ND NI nNN ND NG 101746 NG KDNI NG NG NI KD NI KD KD NG nNN KD NI nNN KD NG 101747 NG cND NI NG NG NIcND NI cND cND NG nNN cND NI nNN cND NG 101748 NG HD NI NG NG NI HD NIHD HD NG SN HD NI SN HD NG 101749 NG HD NI NG NG NI HD NI HD HD NG AK HDNI AK HD NG 101750 NG HD NI NG NG NI HD NI HD HD NG DH HD NI DH HD NG101751 NG HD NI NG NG NI HD NI HD HD NG cHN HD NI cHN HD NG 101752 NG HDNI NG NG NI HD NI HD HD NG KN HD NI KN HD NG 101753 TP HD NI TP TP NI HDNI HD HD TP nNN HD NI nNN HD TP 101754 IA HD NI IA IA NI HD NI HD HD IAnNN HD NI nNN HD IA 101755 VG HD NI VG VG NI HD NI HD HD VG nNN HD NInNN HD VG 101756 SGgs HD NI SGgs SGgs NI HD NI HD HD SGgs nNN HD NI nNNHD SGgs 101757 IP HD NI IP IP NI HD NI HD HD IP nNN HD NI nNN HD IP

TABLE 29C Single RVD substitutions Single 101758 NG HD HI NG NG NI HD NIHD HD NG nNN HD NI nNN HD NG 101759 NG HD KI NG NG NI HD NI HD HD NG nNNHD NI nNN HD NG 101760 NG HD RI NG NG NI HD NI HD HD NG nNN HD NI nNN HDNG 101761 NG ND NI NG NG NI HD NI HD HD NG nNN HD NI nNN HD NG 101762 NGKD NI NG NG NI HD NI HD HD NG nNN HD NI nNN HD NG 101763 NG cND NI NG NGNI HD NI HD HD NG nNN HD NI nNN HD NG 101764 NG HD NI NG NG NI HD NI HDHD NG SN HD NI nNN HD NG 101765 NG HD NI NG NG NI HD NI HD HD NG AK HDNI nNN HD NG 101766 NG HD NI NG NG NI HD NI HD HD NG DH HD NI nNN HD NG101767 NG HD NI NG NG NI HD NI HD HD NG cHN HD NI nNN HD NG 101768 NG HDNI NG NG NI HD NI HD HD NG KN HD NI nNN HD NG 101769 NG HD NI NG TP NIHD NI HD HD NG nNN HD NI nNN HD NG 101770 NG HD NI NG IA NI HD NI HD HDNG nNN HD NI nNN HD NG 101771 NG HD NI NG VG NI HD NI HD HD NG nNN HD NInNN HD NG 101772 NG HD NI NG SGgs NI HD NI HD HD NG nNN HD NI nNN HD NG101773 NG HD NI NG IP NI HD NI HD HD NG nNN HD NI nNN HD NG All typical101041 NG HD NI NG NG NI HD NI HD HD NG nNN HD NI nNN HD NG

These novel TALENs were then tested for cleavage activity against theendogenous CCR5 locus at 30 and 37 degrees, and analyzed by the Cel-Iassay as described previously, and were shown to be active at inducingNHEJ (e.g. see FIG. 30). Note that the unlabeled lane represents anon-functional TALEN construct with a frame shift mutation.

The results show that the novel (atypical) RVDs are capable of cleavingDNA when in used in TALEN proteins in which each TALE-repeat unitincludes a novel RVD, as well as in type substituted or singlysubstituted TALENs.

Example 16 Novel TALE C-Terminal Half Repeats

The majority of natural TALEs use the NG RVD in the C-terminal halfrepeat to specify interaction with a T nucleotide base. Thus, generationof novel C-terminal half repeats was investigated to allow for theexpansion of TALE targeting. TALENs targeting the Pou5F1 and PITX3 geneswere used as backbones, and the RVD within the C-terminal half repeat(C-cap amino acids C-9 and C-8) was altered to specify alternate nucleicacids. In these mutants, the NI RVD was inserted to recognize A, HD forC, NK for G and the control was NG for T. The TALENs used containedbetween 15 and 18 RVDs and targeted a variety of target sequences inthese two genes.

The results are shown in FIG. 29 and demonstrate that the RVD positionin the C-terminal half repeat can be engineered to interact withnucleotide bases other than only T, or can be designed to recognize allbases equally. The lane assignments, target sequences, and % NHEJ asmeasured in this Cel-I assay are shown below in Table 30.

TABLE 30 Novel C-terminal half repeat targets No. SBS# Target Bindingsequence NHEJ % 1 101124 Pou5F1 5′ GCAGCTGCCCAGACCT (SEQ ID 2.2 NO: 185)101126 5′ GACCCTGCCTGCT (SEQ ID NO: 186) 2 101125 Pou5F15′ GACCCTGCCTGCTCCT (SEQ ID 5.0 NO: 187) 101225 5′CACCTGCAGCTGCCCAG (SEQID NO: 188) 3 101139 Pou5F1 5; GGGCTCTCCCATGCAT (SEQ ID 6.7 NO: 189)101141 5′TCCTAGAAGGGCAGGC (SEQ ID NO: 190) 4 101138 Pou5F15′CTGGGCTCTCCCAT (SEQ ID 25.6 NO: 191) 101229 5′CCCCCATTCCTAGAAGG (SEQID NO: 192) 5 101151 Pitx3 5′CCGCACCCCCAGCT (SEQ ID 13.3 NO: 193) 1012335′GCTCCTGGCCCTTGCA (SEQ ID NO: 194) 6 101231 Pitx3 5′GGCACTCCGCACCCCCA(SEQ ID 10.0 NO: 195) 101234 5′ACCGCTGTGCTCCTGGC (SEQ ID NO: 196) 7101230 Pitx3 5′GGCACTCCGCACCCC (SEQ ID 4.9 NO: 197) 1011565′TACCGCTGTGCTCCT (SEQ ID NO: 198) 8 101236 Pitx3 5′ACGCCGTGGAAAGGCC(SEQ ID 2.5 NO: 199) 101237 5′CGGGGATGATCTACGG (SEQ ID NO: 200) 9 101235Pitx3 5′ACGCCGTGGAAAGGC (SEQ ID 8.1 NO: 201) 101238 5′CGGGGATGATCTAC(SEQ ID NO: 202) 10 101236 Pitx3 5′ACGCCGTGGAAAGGCC (SEQ ID 9.2 NO: 203)101238 5′CGGGGATGATCTAC (SEQ ID NO: 204) 11 101167 Pitx35′CGTTGCCCCCGCCCT (SEQ ID 13.1 NO: 205) 101239 5′ATGAGCGGCCCCGCC (SEQ IDNO: 206) 12 101166 Pitx3 5′GAGCGGCCCCGCCCGT (SEQ ID 5.3 NO: 207) 1011675′CGTTGCCCCCGCCCT (SQ ID NO: 208) 13 101239 Pitx3 5′ATGAGCGGCCCCGCC (SEQID 11.2 NO: 209) 101240 5′GAATCGTTGCCCCCGC (SEQ ID NO: 210) 14 101166Pitx3 5′GAGCGGCCCCGCCCGT (SEQ ID 10.7 NO: 211) 101240 5′GAATCGTTGCCCCCGC(SEQ ID NO: 212) This data demonstrates that TALENs with novel halfrepeats are capable of cleaving their respective targets.

Example 17 Identification of Optimal Target Sequences

To determine optimal target sequences, and thus optimal TALEN proteindesign, an in silico analysis was done using the results from multipleSELEX assays to determine i) the best target for the R1 repeat(N-terminal repeat) unit and ii) how specific RVD repeats behave in thecontext of their neighboring repeat units in dimer and trimer settings.In these studies, the NI RVD was used to recognize A, HD for C, NN forG, and NG for T.

Results are summarized in Tables 31, 32 and 33. The values in Table 31are log-odds scores calculated as the logarithm (base 4) of the ratiobetween the observed frequency of the targeted base and the frequency ofthat base expected by chance (i.e. 0.25). A score of 1.0 would indicatethat the targeted base was observed 100% of the time (i.e. 4 times morefrequent than expected by chance), a score of 0.0 would indicate thatthe targeted base was observed 25% of the time, and a negative score,would indicate that the targeted base was observed less than 25% of thetime. The values in Table 31 were calculated from the average basefrequency for the appropriate positions of a data set consisting ofSELEX data from 62 separate TALE proteins. The values labeled “R1 RVD”refer to the N-terminal TALE repeat (and cognate position in eachbinding site). The values labeled “R2+RVD) refer to all other RVDs (andcognate positions in each binding site). This data indicates a dramaticdifferent in the specificity of TALE repeats bearing HD, NN, and NG RVDsat the N-terminal position versus all other positions.

The values shown in Tables 32 and 33 represent the change in thoselog-odds scores determined for each base independently versus the scorein either the dimer (Table 32) or trimer (Table 33) setting and weredetermined from SELEX data for 67 separate TALE proteins. Thus the −0.12value for an NN RVD adjacent to an HD RVD (with the NN RVD closer to theN-terminus of the construct and the HD RVD closer to the C-terminus ofthe construct) indicates that the sum of the log-odds scores for bothpositions in the dimer was 0.12 less than would be expected if these twoRVDs behaved independently of each other. Similarly, the −0.34 value inTable 33C indicates that an NN RVD flanked on the N-terminal side by asecond NN RVD and flanked on the C-terminal side by an HD RVD indicatesthat the NN RVD of interest has a log-odds score 0.34 less than theaverage value for all NN RVDs. In Tables 32, 33A, 33B, 33C, and 33D,negative values indicate combinations of adjacent RVDs that perform morepoorly than if they were completely independent of each other.

TABLE 31 Log-odds scores for RVD specificity at single positions R1 RVDR2 + RVD NI (A) 0.87 NI (A) 0.88 HD (C) 0.39 HD (C) 0.89 NN (G) 0.42 NN(G) 0.71 NG (T) 0.31 NG (T) 0.85

TABLE 32 Change in log-odds scores for RVD specificity for two adjacentRVDs C-terminal RVD → RVD NI (A) HD (C) NN (G) NG (T) N-terminal NI (A)0.03 0.07 −0.10 0.11 RVD HD (C) 0.04 0.04 −0.05 −0.04 ↓ NN (G) 0.12−0.12 −0.08 0.07 NG (T) 0.07 −0.10 0.15 −0.20

TABLE 33A Change in log-odds scores for RVD specificity in trimerpositions, NI (A) in middle C-terminal RVD → RVD NI (A) HD (C) NN (G) NG(T) N-terminal NI (A) 0.06 0.11 −0.06 0.04 RVD HD (C) 0.03 0.00 −0.060.02 ↓ NN (G) −0.03  0.06 −0.01 0.02 NG (T) −0.03  0.05  0.07 −0.05

TABLE 33B Change in log-odds scores for RVD specificity in trimerpositions, HD (C) in middle C-terminal RVD → RVD NI (A) HD (C) NN (G) NG(T) N-terminal NI (A) 0.02 0.03 0.06 −0.01 RVD HD (C) 0.05 0.02 0.090.00 ↓ NN (G) 0.07 0.04 0.07 −0.01 NG (T) 0.04 −0.01 −0.51  −0.17

TABLE 33C Change in log-odds scores for RVD specificity in trimerpositions, NN (G) in middle C-terminal RVD → RVD NI (A) HD (C) NN (G) NG(T) N-terminal NI (A) 0.07 −0.23 0.04 −0.03 RVD HD (C) 0.20  0.04 0.200.09 ↓ NN (G) −0.12  −0.34 0.01 −0.01 NG (T) 0.15 −0.17 0.13 0.12

TABLE 33D Change in log-odds scores for RVD specificity in trimerpositions, NG (T) in middle C-terminal RVD → RVD NI (A) HD (C) NN (G) NG(T) N-terminal NI (A) 0.11 0.10 0.14 0.08 RVD HD (C) ND −0.07 0.07 −0.11↓ NN (G) 0.09 −0.12 0.05 −0.01 NG (T) 0.04 −0.07 −0.05 −0.27Note: in Tables 33A through 33D, italics indicate less than 3 values inthe dataset, where all other numbers contain at least 3 values used fordetermining the probability changes.

These results demonstrate that there is context dependency for optimalrepeat unit binding, and indicate that for optimal protein design/targetidentification, the repeat units are not completely modular. As a whole,these data can be used to propose design rules to optimize both thetarget selection for a particular TALE and for designing the optimalTALENs. For example, it appears that NI is the least context dependentRVD and the best RVD at the R1 position is NI (e.g. ideally target sitesshould start with TA to accommodate R0 and R1-NI). It appears that AC,AT, CC, CA, TA, AA are the best dimers to target while GG, GC, AG, TT,CG, GT, and TC are the worst. In terms of triplets, AAC, ATG, GCA, ATA,ACG, and ATC are very good triplets to target while GGC, AGC, TGC, TTT,GGA, AGT, GGT, GGG, TCT, GTC, CTT, and AGG appear to be the worst. Thus,these design rules can be combined to create the optimally bindingTALENs. Similarly, SELEX studies with NK, AK, and DK RVDs in Table 28and additional SELEX studies with NK RVDs (FIG. 17A) indicate that RVDswith lysine (K) at position 13 tend to cause adjacent NI RVDs C-terminalto the NK, AK, or DK RVD to specify G rather than A. Thus design rulesdetermined for typical RVDs and the NK RVDs should also apply toatypical RVDs with the same residue at position 13.

Example 18 Demonstration of TALEN-Driven Targeted Integration in HumanStem Cells

To demonstrate the versatility of the TALEN system, TALENs were used todrive targeted integration in human embryonic stem cells (ESC) andinduced pluripotent stem cells (iPSC). Human ESCs and iPSCs were usedfor the targeted integration of a puromycin donor nucleic acidadditionally comprising a restriction site, into the AAVS1 locus whereexpression of the puromycin marker is driven by the AAVS1 promoter.Donors and methods followed were those described previously in co-ownedWO2010117464 (see also Hockemeyer et al (2009) Nat Biotechnol 27(9):851-857, in which we demonstrated that the spontaneous frequency oftargeted integration of such a construct into the AAVS1 locus is belowthe limit of detection of our assay). Nucleases used were TALENsspecific for the AAVS1 locus as described in Example 11, and the targetbinding site is shown below:

101077 TCCCCTCCACCCCACAGT ggggccactagggacAGGATTGGTGACAGAAAA (SEQ ID NO:213) AGGGGAGGTGGGGTGTCAccccggtgatccctg TCCTAACCACTGTCTTTT (SEQ ID NO:214)                        101079

First, this locus was targeted with a gene trap approach in which thepuromycin resistance gene (PURO) was expressed under the control of theendogenous PPP1R12C promoter only following a correct targeting event.Second, the PPP1R12C locus was targeted using an autonomous selectioncassette that expressed the puromycin resistance gene PURO from thephosphoglycerate kinase (PGK) promoter. Clones of puromycin resistantcells were grown and screened by Southern blot against restricted DNAusing standard methods. The probe used in this experiment was againstthe PPP1R12C/AAVS1 locus and recognized a sequence that is the smallrestriction fragment of DNA (and thus had a higher mobility) withincorporated donor. Targeting efficiency was high independent of thedonor used, with approximately 50% of isolated clones possessing eitherheterozygous or homozygous correctly targeted events and carrying thetransgene only at the desired locus. This efficiency is comparable tothat previously observed with ZFNs. Targeting to the PPP1R12C locusresulted in expression of the introduced transgene. Uniform expressionof enhanced green fluorescent protein (eGFP) was observed in hESCs andiPSC when targeted with the SA-PURO donor plasmids that additionallycarries a constitutive eGFP expression cassette. Importantly, hESCs thathave been genetically engineered using TALENs remained pluripotent asindicated by their expression of the pluripotency markers OCT4, NANOG,SSEA4, Tra-1-81 and Tra-1-60.

TALENs were also designed against the first intron of the human OCT4gene (OCT4-Intl-TALEN) and the target sequence is shown below incombination with three different donor plasmids:

101125: GACCCTGCCTGCTCCT (SEQ ID NO: 329) 101225: CACCTGCAGCTGCCCAG (SEQID NO: 330)The TALENs utilized a +63 C-cap and used the typical RVDs (NI, HD, NN,and NG to target A, C, G, and T respectively). 101125 comprised 15.5TALE repeats and 101225 comprised 16.5 TALE repeats. 101225 utilized ahalf repeat with an NN RVD to recognize the 3′ G in its target site.

Correct targeting events are characterized by expression of bothpuromycin and an OCT4 exon1-eGFP fusion protein under control of theendogenous OCT4 promoter. The first two donor plasmids were designed tointegrate a splice, acceptor eGFP-2A-self-cleaving peptide(2A)-puromycin cassette into the first intron of OCT4, and differedsolely in the design of the homology arms, while the third donor wasengineered to generate a direct fusion of exon 1 to the reading frame ofthe eGFP-2A-puromycin cassette. Both strategies resulted in correcttargeted gene addition to the OCT4 locus as determined by Southern blotanalysis and DNA sequencing of single-cell-derived clones. Targetingefficiencies ranged from 67% to 100% in both hESCs and iPSCs.

To test whether TALENs can be used to genetically engineer loci that arenot expressed in hESCs, TALENs were engineered (using the same designand assembly procedure used for 101125 and 101225) to cleave within thefirst coding exon of the PITX3 gene. The target sequences are shownbelow:

101148: GGCCCTTGCAGCCGT (SEQ ID NO: 331) 101146: CAGACGCTGGCACT (SEQ IDNO: 332)

After electroporation, targeting events were evaluated by Southern blotanalysis using an external 5′ and an internal 3′ probe.Single-cell-derived clones carrying the donor-specified eGFP transgenesolely at PITX3 were obtained on average 6% of the time. Of note, one of96 hESC clones analyzed carried the transgene on both alleles of PITX3Exon1 (in WI#3) hESCs demonstrating the successful genetic modificationof both alleles of a non-expressed gene in a single step.

These results demonstrate the ability to use TALENs to drive targetedintegration into the genome of stem cells.

Example 19 Examples of TALEN Mediated Gene Editing In Vivo

TALEN genome editing in C. elegans. To demonstrate that TALENs could beused in animals for in vivo gene editing, the following experiments wereconducted. A TALEN pair specific for the Caenorhabditis elegans ben-1mutation were delivered as RNA and screened for benomyl resistance asdescribed in Driscoll et al ((1989) J. Cell. Biol. 109:2993-3003). Theben-1 mutant phenotype is dominant and visible in 100% of progeny undera regular dissecting microscope. Briefly, wild-type C. eleganshermaphrodites were reared on regular NGM agar plates before injectionwith mRNAs encoding TALENs targeting ben-1.

The nucleic acids encoding the TALENs were inserted into an SP6 in vitrotranscription vector (IVT) using standard restriction cloningprocedures. The ICT vector backbone was derived from pJK370 and contains5′ and 3′ UTR sequences shown previously to support germ-linetranslation (see Marin and Evans (2003) Development 130: 2623-2632).Production of mRNAs containing 5′ CAP structures and poly A wasperformed in vitro using the mMessage mMachine® (Ambion) and polyAtailing kits (Ambion) and purified over a Ambion MEGAClear™ column priorto quantitation using a NanoDrop spectrophotometer (Thermoscientific).mRNA injections were performed under a Zeiss Axiovert microscope using aNarishige IM300 injector. Injection of mRNAs were performed according tostandard C. elegans DNA injection protocols (see Stinchcomb et al.(1985) Mol Cell Biol 5:3484-3496) with the following differences: theregulator was adjusted such that the pressure from the N2 gas tank was60 psi. The P_(inject) and P_(balance) measurements were adjusted to 15psi and 2 psi, respectively. These pressure values are lower than thosetypically used for DNA injections to allow a more gentle release offluid into the worm gonad. All mRNAs were injected at 500 ng/μL, and allmRNAs encoding the TALENs were injected as pairs, thus the total mRNAconcentration in the needle was 1000 ng/μL.

Following mRNA injection, the animals were transferred to platescontaining 7 μM benomyl. F1 self-progeny were screened as young adultsby touching the anterior side of the animal. Heterozygous mutant animalsrespond by reversal using multiple sinusoidal-like movements, whereaswild-type animals are paralyzed and lack this ability. Non-paralyzed F1animals were either lysed individually for PCR/Cel-I analysis of thetarget site (as described above), or transferred individually to freshbenomyl plates and homozygotes isolated from non paralyzed F2 bysequencing over the target site. One TALEN pair, designated101318/101321, caused reversion of the ben-1 mutation phenotype, and theF1 progeny were found to be resistant to benomyl. Sequence analysis ofthe benomyl resistant animals revealed two different bona fide indels atthe target location. The locus in the target site for this TALEN pair isshown below, and their sequences are shown in Example 23.

101318 TCCAGCCTGATGGAAC ttataagggagaaagtgATTTGCAGTTGGAAAGAA  (SEQ ID NO:215) AGGTCGGACTACCTTGaatattccctctttcac TAAACGTCAACCTTTCTT   (SEQ ID NO:216)                                                     101321

These data demonstrate that TALENs are capable of genomic editing invivo.

TALEN genome editing in rats. Next, TALENs were used to edit the ratgenome. The rat IgM-specific TALEN pair 101187/101188 that targets Exon2 in the endogenous rat IgM gene was constructed as previously describedin Examples 11 and 12 above. The target sequence in the rat genome isshown below where the bold and upper case letters indicate the targetsite for the TALE DNA binding domain and the lowercase letters indicatethe gap or spacer region:

     101187SEQ ID 380: 5′-TTCCTGCCCAGCTCCATttccttctcctggaactACCAGAACAACACTGAA -3′ SEQ ID 381: 3′-AAGGACGGGTCGAGGTAaaggaagaggaccttgaTGGTCTTGTTGTGACTT -5′                                                    101188

Nucleic acids encoding these TALEN pair were then injected into ratembryos as described in Menoret et al (2010) Eur J Immunol. October;40(10): 2932-41. Nucleic acids encoding the TALENs were injected eitheras a pronuclear (PNI, DNA) or an intracytoplasmic (IC, RNA) injection atthe doses shown below in Table 35.

TABLE 34 Route and Dose of rat IgM-specific TALENs No. No. Injected/ No.Route/Dose Injected Transfered Transfered No. mutant StrainTarget/Construct (ng/μl) embryos embryos (%) No. pups founders foundersSD IgM/TALEs PNI/10 166 98 59.04 13  13 3 SD IgM/TALEs PNI/2 236 15063.56 53  53 4 SD IgM/TALEs PNI/0.4 84 59 70.24 3 + 6 ND ND transferredmothers* SD IgM/TALEs IC/10 200 141 70.5 6 ND ND transferred mothers* SDIgM/TALEs IC/4 187 122 65.2 7 ND ND transferred mothers* SD IgM/TALEsIC/0.8 184 143 77.7 6 ND ND transferred mothers* *Note: not allexpectant mothers had delivered, ND = not determined

A percentage of the injected embryos were implanted into pseudo pregnantfemale rats and resultant newborns were assayed for genome editing. DNAwas isolated from the pups resulting from the pronuclear DNA injectionsand subjected to a T7 mismatch analysis as described in Kim et al (2009)Genome Res. 19(7): 1279-1288. Briefly, PCR was performed using theprimer set GJC153F-154R to create a 371 bp PCR product. The primer pairis shown below:

GJC 153F primer: (SEQ ID NO: 453) 5′ ggaggcaagaagatggattc GJC 154Rprimer: (SEQ ID NO: 454) 5′ gaatcggcacatgcagatct

For this analysis, 100 ng tail gDNA was used which had been isolated bystandard practice. Potential heteroduplexes were allowed to form using 5ul of the PCR product as follows: 2′ at 95° C./95° C. to 85° C. (−2°C./sec)/85° C. to 25° C. (−0.1° C./sec)/4° C. This was then digestedwith T7 endonuclease I (NEBiolabs ref: M0302L) under the followingconditions: 5 ul PCR heteroduplex+1 ul 10×NEB2+0.5 ul T7 endo+3.5 ulH2O/20′ a 37° C. Following digestion, the reaction was run on a 1.2%agarose gel in 0.5×TAE. 7 of the 66 pups analyzed were positive for NHEJactivity by the T7 assay, (shown in FIG. 31) and sequencing revealed thepresence of a NHEJ associated indels (e.g. 1 bp deletion in rat 3.3 anda 90 bp deletion in rat 3.4).

TALEN pairs are also used for targeted integration with a nucleic acidof interest into rat cells to generate transgenic animals. The rat cellstargeted by the TALEN pair are rat embryonic stem cells, one- ormore-celled GFP-containing rat embryos or any rat cell type convertibleto an induced pluripotent stem (iPS) cell. The TALEN pair is deliveredto the cell and can be plasmid DNA, optimally containing a CAG promoter,mRNA, optimally with a 5′ cap structure and a 3′ poly-adenosine tail,purified protein or viral particles containing nucleic acid encoding theTALEN open reading frames. The donor DNA can single- or double-strandedcircular plasmid DNA containing 50-1000 bp of homology on both sides ofthe break site or single- or double-stranded linear plasmid DNAcontaining 50-1000 bp of homology on both sides of the break site. TheTALEN and donor are delivered by microinjection of rat cells or embryos,transfection of rat cells via electroporation, lipid-based membranefusion, calcium phosphate precipitation, PEI, etc., incubation withpurified nuclease protein (for example, if fused to a cell-penetratingpeptide), or infection of rat cells or embryos with a virus. Thesemethods are known in the art. The means of generating a modified ratfrom the injected or transfected cells or embryos will depend on thedelivery method chosen. For embryos, the embryos will be implanted intothe uterus of a pseudo-pregnant rat and allowed to come to term asdescribed previously. For modified cells, three methods are possible: a)if the rat cells are embryonic stem cells, rat blastocysts should beinjected with the modified rat stem cells. Blastocysts will be implantedinto the uterus of a pseudo-pregnant rat and allowed to come to term; b)the cell (or its nucleus) should be microinjected into an enucleatedoocyte (somatic cell nuclear transfer) and the resulting embryoimplanted into the uterus of a pseudo-pregnant rat and allowed to cometo term or c) the cell should be converted to an iPS cell and should beinjected into a rat blastocyst. Blastocysts will be implanted into theuterus of a pseudo-pregnant rat and allowed to come to term. Pups arethen assayed for presence of the transgene by PCR or any other meansknown in the art.

TALEN genome editing in plants. TALEN pairs specific for the Z. maizeRPD1 and C1 genes were constructed as described above in Example 11 andtheir target sequences are shown below in comparison with the RPD1 locus(SEQ ID NOs: 382 through 387):

TTATTTGAAGAAACTAT                (101389)TTATTTGAAGAAACT                  (101388)   TTTGAAGAACTATATT              (101390)TTATTTGAAGAAACTATATTACAGAGCATAAGCTTATGCAACACTCCCACTAGTTGATTAATAAACTTCTTTGATATAATGTCTCGTATTCGAATACGTTGTGAGGGTGATCAACTAA                                   TACGTTGTGAGGGT            (101391)                                       TTGTGAGGGTGATCAAGT    (101393)

TALEN pairs made against the C1 locus are similarly shown below, (SEQ IDNOs: 388 through 390):

TGGGGAGGAGGGCGTGCT                      (101370)TGGGGAGGAGGGCGTGCTGCGCGAAGGAAGGCGTTAAGAGAGGGGCGTGGACGAGCAAGGACCCCTCCTCCCGCACGACGCGCTTCCTTCCGCAATTCTCTCCCCGCACCTGCTCGTTCC                                    TCTCTCCCCGCACCTGCT (101371)

Additional TALEN pairs were made against the C1 locus as follows, (SEQID NOs: 391 through 398):

 TGAACTACCTCCGGCCC                                                (101378)                                      TCCTACGACGAGGAGGAT          (101380)CTGAACTACCTCCGGCCCAACATCAGGCGCGGCAACATCTCCTACGACGAGGAGGATCTCATGATCATCCGCCTGACTTGATGGAGGCCGGGTTGTAGTCCGCGCCGTTGTAGAGGATGCTGCTCCTCCTAGAGTACTAGTAGGCGGA                                    TAGAGGATGCTGCTCCT              (101379)CCACAGGCTCCTCGGCAACAGGT GGTGTCCGAGGAGCCGTTCTCCA  TGTCCGAGGAGCCGTT    (101381)

The plant specific TALEN pairs were analyzed in mammalian Neuro 2A cellsfor activity using the Dual-Luciferase Single Strand Annealing Assay(DLSSA). This is a novel system used to quantify ZFN or TALEN activitiesin transiently transfected cells, and is based on the Dual-LuciferaseReporter® Assay System from Promega. See, Example 13. The system allowsfor sequential measurement of two individual reporter enzymes, Fireflyand Renilla Luciferases, within a single tube (well). Both of theFirefly and the Renilla Luciferase reporters are re-engineered and theassay conditions are optimized. The Firefly Luciferase reporterconstruct contains two incomplete copies of the Firefly coding regionsthat are separated by DNA binding sites for either ZFNs or TALENs. Inthis study, the 5′ copy is derived from approximately two third of theN-terminal part of the Firefly gene and the 3′ copy is derived fromapproximately two third of the C-terminal part of the Firefly gene. Thetwo incomplete copies contain about 600-bp homology arms. The separatedFirefly fragments have no luciferase activity. A DNA double strand breakcaused by a ZFN or TALEN pair will stimulate recombination betweenflanking repeats by the single-strand annealing pathway and then restorethe Firefly luciferase function. The co-transfected Renilla Luciferaseplasmid provides an internal control. The luminescent activity of eachreporter is read on a luminometer. Normalizing the activity of theexperimental reporter (Firefly) to the activity of the internal control(Renilla) minimizes experimental variability caused by differences incell viability and/or transfection efficiency. The normalized value isused to determine the activity of a given ZFN or TALEN pair. This is auseful tool when working in systems with precious model cells or whenthe intended target cell type is either not available or difficult to beused for screening purpose. This is also useful tool to develop and tooptimize TALEN technology platform when the target sequences are notavailable in endogenous genome. Active nucleases can be identified byDLSSA and then ported into the endogenous system for final evaluation.The active TALEN pairs on the plant targets are shown below in Table35A.

TABLE 35A Plant TALENs PAIR TARGET T1 T2 Activity* 1 C1 101370 1013715.0 2 C1 101378 101379 7.1 3 C1 101380 101381 10.3 4 RPD1 101388 1013917.6 5 RPD1 101389 101391 7.2 6 RPD1 101389 101393 9.9 7 RPD1 101390101391 9.7 8 RPD1 101390 101393 9.6 Control CCR5 41 47 12.0 Control pVax0.2 *Note: Activity in this assay is measured in relative units in theluciferase SSA assay.

The TALEN pairs were then delivered via gold-particle bombardment tomaize Hi II embryos using standard methods (Frame et al, (2000) In vitrocellular & developmental biology. 36(1): 21-29). In total, approximately90 pollinated maize embryos per TALEN pair were transformed and allowedto grow for ca. seven days on callus initiation media prior to poolingand freezing in liquid N2 for genomic DNA extraction. Genomic DNA wasisolated from 4-6 frozen embryos per bombarded plate using the DNeasyPlant Miniprep kit (Qiagen). Each TALEN target was then amplified bytwo-step PCR using High-Fidelity Phusion Hot Start II Polymerase (NEB)from pooled genomic DNA consisting of three biological triplicates. Inthe first round, each site was amplified in a 20-cycle PCR using 400 nggenomic DNA and the primers listed in Table 35B. In the second round, anadditional 20 cycles were performed using 1 ul of product from the firstPCR round and the primers SOLEXA-OUT-F1 and SOLEXA-OUT-R1 to generatecomplete Illumina sequencing amplicons. The resulting PCR products werethen purified on Qiaquick PCR Purification columns (Qiagen), normalizedto 50 nM each, and combined in equal volumes so that a total of eightsites were sequenced in a single Illumina lane. Control amplicons fromuntreated genomic DNA were submitted in a separate lane. Illuminasingle-read 100 bp sequencing was performed at ELIM Biopharmaceuticals(Hayward, Calif.).

TABLE 35B Sequences of oligonucleotide primers used for Illuminasequencing C1.70-71.F1CTACACTCTTTCCCTACACGACGCTCTTCCGATCTggagcttgatcgacgaga (SEQ ID NO: 426)C1.78-79.F2 CTACACTCTTTCCCTACACGACGCTCTTCCGATCTctgtggaggcggatgat (SEQ IDNO: 427) C1.80-81.F1CTACACTCTTTCCCTACACGACGCTCTTCCGATCTactacctccggcccaac (SEQ ID NO: 428)RPD1.88.91.F1CTACACTCTTTCCCTACACGACGCTCTTCCGATCTGGCCgctgcagactctatctcacc (SEQ ID NO:429) RPD1.89.91.F1CTACACTCTTTCCCTACACGACGCTCTTCCGATCTTTCCgctgcagactctatctcacc (SEQ ID NO:430) RPD1.89.93.F1CTACACTCTTTCCCTACACGACGCTCTTCCGATCTCCGGgctgcagactctatctcacc (SEQ ID NO:431) RPD1.90.91.F1CTACACTCTTTCCCTACACGACGCTCTTCCGATCTAACCgctgcagactctatctcacc (SEQ ID NO:432) RPD1.90.93.F1CTACACTCTTTCCCTACACGACGCTCTTCCGATCTCCAAgctgcagactctatctcacc (SEQ ID NO:433) C1.70-71.R1 CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTttccctccatttgccttc(SEQ ID NO: 434) C1.78-79.R2CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTgtgtgtgggtgcaggttt (SEQ ID NO: 435)C1.80-81.R1 CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTtcgtcgtcagctcgtgta (SEQ IDNO: 436) RPD1.88.91.R1CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTtgccaggaacactttcca (SEQ ID NO: 437)RPD1.89.91.R1 CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTtgccaggaacactttcca (SEQID NO: 438) RPD1.89.93.R1CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTtgccaggaacactttcca (SEQ ID NO: 439)RPD1.90.91.R1 CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTtgccaggaacactttcca (SEQID NO: 440) RPD1.90.93.R1CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTtgccaggaacactttcca (SEQ ID NO: 441)SOLEXA-OUT- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACG (SEQ ID F1 NO:442) SOLEXA-OUT- CAAGCAGAAGACGGCATA (SEQ ID NO: 443) R1

The sequencing revealed the presence of numerous indels in the cellpools from the TALEN treated embryos as is shown below in Table 36. Thedetails of the sequence analysis are as follows: barcoded sequencesderived from TALEN treated Zea maize embryos were pooled together andsubmitted for 100 bp read-length sequencing on an Illumina GA2sequencer. Barcoded sequences derived from mock treated Zea maizeembryos were pooled together and submitted for 100 bp read-lengthsequencing on a separate lane of the same Illumina GA2 sequencer.Sequences in each resultant data file were separated by barcode andaligned against the unmodified genomic sequence. A small fraction of theembryos contained a 3 bp insertion in the C1 gene relative to themajority of the embryos. Indels consisting of at least 2 contiguousinserted or deleted bases within a 10 bp window centered on the expectedTALEN cleavage sites were considered potential NHEJ events and wereprocessed further. InDels that occurred with similar frequency in both agiven TALEN treated sample and the cognate mock treated sample wereconsidered sequencing artifacts and were discarded.

TABLE 36 InDels in TALEN treated maize TALEN treated Mock Treated TargetTotal Total gene TALEN pair reads InDel % InDel reads InDel % InDel S1C1 101370/101371 2033338 185 0.0091 1377048 0 0.0000 S2 C1 101378/1013792208608 228 0.0103 2332142 2 0.0001 S3 C1 101380/101381 2213631 3600.0163 2020763 1 0.0000 S4 RPD1 101388/101391 2798647 341 0.0122 26795543 0.0001 S5 RPD1 101389/101391 2823653 414 0.0147 2549110 0 0.0000 S6RPD1 101389/101393 2740241 239 0.0087 2783422 3 0.0001 S7 RPD1101390/101391 2826655 495 0.0175 2790561 0 0.0000 S8 RPD1 101390/1013932601239 482 0.0185 2910777 0 0.0000

Table 37 shows the most observed indels in the eight samples shownabove, demonstrating that the TALENs were capable of inducing NHEJ withboth gene targets and all pairs of nucleases. For each sample, theunaltered genomic sequence is shown with the gap between the two TALENbinding sites underlined. Deleted bases are indicated by colons andinserted bases are indicated by curved brackets with “{” indicating thestart of the inserted sequence and “}” indicating the end of theinserted sequence.

TABLE 37 InDels observed in maize samples S1 TALEN Treated (Gene Target:C1, TALEN pair 101370/101371)GAGCGCGATGGGGAGGAGGGCGTGCTGCGCGAAGGAAGGCGTTAAGAGAGGGGCGTGGACGAGCAAGGAGGAGCGCGATGGGGAGGAGGGCGTGCTGCGCGA:::::::CGTTAAGAGAGGGGCGTGGACGAGCAAGGAGGAGCGCGATGGGGAGGAGGGCGTGCTGCGCGAAGGA::GCGTTAAGAGAGGGGCGTGGACGAGCAAGGAGGAGCGCGATGGGGAGGAGGGCGTGCTGCGCGAA:::AGGCGTTAAGAGAGGGGCGTGGACGAGCAAGGAGGAGCGCGATGGGGAGGAGGGCGTGCTGCGCGA:::::GGCGTTAAGAGAGGGGCGTGGACGAGCAAGGAGGAGCGCGATGGGGAGGAGGGCGTGCTGtGCGA:::AAGGCGTTAAGAGAGGGGCGTGGACGAGCAAGGAGGAGCGCGATGGGGAGGAGGGCGTGCTGCGCGAAGG::::CGTTAAGAGAGGGGCGTGGACGAGCAAGGAGGAGCGCGATGGGGAGGAGGGCGTGCaGCGCG:::::AGGCGTTAAGAGAGGGGCGTGGACGAGCAAGGAGGAGCGCGATGGGG:::::::::::::::::::AGGAAGGCGTTAAGAGAGGGGCGTGGACGAGCAAGGAGS2 TALEN Treated (Gene Target: C1, TALEN pair 101378/101379)GAGATCCTCCTCGTCGTAGGAGATGTTGCCGCGCCTGATGTTGGGCCGGAGGTAGTTCAGCCACCGCAGCGAGATCCTCCTCGTCGTAGGA:::::::::::::::GATGTTGGGCCGGAGGTAGTTCAGCCACCGCAGCGAGATCCTCCTCGTCGTAGGAGATGTTG::::::CTGATGTTGGGCCGGAGGTAGTTCAGCCACCGCAGCGAGATCCTCCTCGTCGTAGGAGATGTTGC::CGCCTGATGTTGGGCCGGAGGTAGTTCAGCCACCGCAGCGAGATCCTCCTCGTCGTAGGAGATGTTGCCGC::CTGATGTTGGGCCGGAGGTAGTTCAGCCACCGCAGCGAGATCCTCCTCGTCGTAGGAGATGTTGC::::::TGATGTTGGGCCGGgGGTAGTTCAGCCACCGCAGCGAGATCCTCCTCGTCGTAGGAGATGTTG:::::::::::::::GGCCGGAGGTAGTTCAGCCACCGCAGCGAGATCCTCCTCGTCGTAGGAGATGTTGCCGCG::::ATGTTGGGCCGGAGGTAGTTCAGCCACCGCAGCGAGATCCTCCTCGTCGTAGGAGATGTT:::::GCCTGATGTTGGGCCGGAGGTAGTTCAGCCACCGCAGCGAGATCCTCCTCGTCGTAGGAGATGTTGCC::::CTGATGTTGGGCCGGAGGTAGTTCAGCCACCGCAGCGAGATCCTCCTCGTCGTAGGAGATGTTGCC::::::GATGTTGGGCCGGAGGTAGTTCAGCCACCGCAGCS3 TALEN Treated (Gene Target: C1, TALEN pair 101380/101381)GGCAACATCTCCTACGACGAGGAGGATCTCATCATCCGCCTCCACAGGCTCCTCGGCAACAGGTCGGTGCGGCAACATCTCCTACGACGAGGAGGATCTCATC:::::CCTCCACAGGCTCCTCGGCAACAGGTCGGTGCGGCAACATCTCCTACGACGAGGAGGATCTCATC::::GCCTCCACAGGCTCCTCGGCAACAGGTCGGTGCS4 TALEN Treated (Gene Target: RPD1, TALEN pair 101388/101391)CTCGGAAGTTATTTGAAGAAACTATATTACAGAGCATAAGCTTATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATTACAGAGCATA{TA}AGCTTATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATTA:::::::::AGCTTATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATTACAGAGCA::AGCTTATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATTACAG::::::::CTTATGCAACACTCCCACTAGTTCATTTTTCTCGG::::::::::::::::::::::::::::::::AAGCTTATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATAT:::::::::::::::::::CAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATT:::::GC:::AGCTTATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTg:::::::::::::::::::::::GCATAAGCTTATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACT:::::::::::::::::::TATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATTACAGAGCA::::::::::::ACACTCCCACTAGTTCATTTTTS5 TALEN Treated(Gene Target: RPD1, TALEN pair 101389/101391)CTCGGAAGTTATTTGAAGAAACTATATTACAGAGCATAAGCTTATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATTACA:::::::AGCTTATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATTACAGA:::::AGCTTATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATTACAGAGC::::::TTATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATTACAG::CATAAGCTTATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATTACAG::::TAAGCTTATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATTAC::::::TAAGCTTATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATTACA::::::AAGCTTATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATTACAGA:::TAAGCTTATGCAACACTCCCACTAGTTCATTTTTCTCGG::::::::::::::::::::::::::::::::AAGCTTATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATTAa:::::::AAGCTTATGCAACACTCCCACTAGTTCATTTTTS6 TALEN Treated (Gene Target: RPD1, TALEN pair 101389/101393)CTCGGAAGTTATTTGAAGAAACTATATTACAGAGCATAAGCTTATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATTACc::::::::::::ATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATT::::::::::::::TATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATTAC::::::::::CTTATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATAT::::::::::::::::::GCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATTA:::::::::::::::TGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATTACA:::::::::::TATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATTACAGAGCATA::CTTATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATTACAGA:::::::::::TGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATTACAGAGCATA:::TTATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATTACAa::::::::::TATGCAACACTCCCACTAGTTCATTTTTS7 TALEN Treated (Gene Target: RPD1, TALEN pair 101390/101391)CTCGGAAGTTATTTGAAGAAACTATATTACAGAGCATAAGCTTATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATTACAGAG::::AGCTTATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATTACAGA:::::AGCTTATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATTACAGAGC::::::TTATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATTACAGAG:::AAGCTTATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATTACAGAGCA:::GCTTATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATTACAGAGCA::AGCTTATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATTACAGA::::AAGCTTATGCAACACTCCCACTAGTTCATTTTTCTCGG::::::::::::::::::::::::::::::::AAGCTTATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATTACAGAGC::::GCTTATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATTACAGAG:::::GCTTATGCAACACTCCCACTAGTTCATTTTTS8 TALEN Treated(Gene Target: RPD1, TALEN pair 101390/101393)CTCGGAAGTTATTTGAAGAAACTATATTACAGAGCATAAGCTTATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATTACAGAGC::::::TTATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATTACAGAGCA::AGCTTATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATTACAGAGCA:::GCTTATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATTACAGAGC::::GCTTATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATTACAGAGCA::::CTTATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATTACAG::::::::::TATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATTACAGAGC:::::::TATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATTACAGA:::::::::TATGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACTATATTA:::::::::::::::TGCAACACTCCCACTAGTTCATTTTTCTCGGAAGTTATTTGAAGAAACT:::::::::::::::::::TATGCAACACTCCCACTAGTTCATTTTT

The InDel frequency was similar in all samples (from 0.0087% to 0.0185%or about 1 in 11,000 events to 1 in 5,400 events). This implies that thelimiting factor is the biolistic delivery to the maize embryos ratherthan the TALEN activity. Barcoded sequences derived from TALEN treatedZea maize embryos were pooled together and submitted for 100 bpread-length sequencing on an Illumina GA2 sequencer.

Next, these TALENs are used to drive targeted integration (TI) of anydesired DNA of interest into the DSB created by TALENs. TI can beaccomplished in monocots or dicots using methods known in the art (seefor example Shukla et al (2009) Nature 459:437 and Cai et al (2009)Plant Mol Biol 69:699). Novel plant species may also be generated stablytransgenic for a selected TALEN as desired, allowing crossing of theTALEN strain to another in which a mutation is desired, followed bysegregation of progeny such that some progeny contain only the desiredmutation and the TALEN transgene has been segregated away.

Thus, these examples demonstrate that the novel TALENs of the inventionare capable of genomic editing in vivo in plant and animal systems.

Example 21 Alterations of the TALE Repeat Unit

To explore alterations in the TALE repeat unit, sequence from bothXanthomonas and Ralstonia were compared. 52 unique repeat units fromRalstonia were examined to observe residue frequencies at each location,and then these values were compiled. The data are presented below inTable 38 where the amino acids are indicated in one letter code fromleft to the right and the position on the repeat unit is indicated fromtop to the bottom, and the RVD positions are indicated in bold:

TABLE 38 Frequencies of amino acids found in Ralstonia repeats A C D E FG H I K L M N P Q R S T V W Y 1 52 2 1 7 40 4 3 2 4 45 1 4 25 24 3 5 526 1 3 48 7 1 51 8 50 2 9 44 8 10 52 11 1 51 12 1 25 18 2 6 13 12 3 6 1 319 3 2 2 1 14 52 15 52 16 50 1 1 17 1 9 41 1 18 52 19 52 20 1 51 21 47 23 22 5 47 23 1 1 2 7 15 17 1 8 24 43 2 2 1 1 3 25 4 4 7 10 2 25 26 8 431 27 21 19 1 8 3 28 10 13 8 2 2 1 16 29 51 1 30 1 51 31 27 21 2 2 32 423 7 33 51 1 34 2 50 35 27 3 5 14 1 1 1

These repeat units then can be combined with those from Xanthamonas tocreate unique repeat units. Repeat sequences that are combinations ofresidues found in Ralstonia repeats and residues found in Xanthomonasresidues could yield proteins with improved properties such as increasedDNA binding affinity, increased DNA binding specificity, or decreasedsensitivity to oxidation. Examples of such repeat unit combinationsinclude, with altered residues indicated in bold and a larger font size:

(SEQ ID NO: 333) LTPDQVVAIASHDGGKQALETVQRLLPVLCQDHG Current Xanthomonas(SEQ ID NO: 334) LTPDQVVAIASHDGGKQALEAVRALLPVLCQDHG Hybrid1 (SEQ ID NO:335) LTPDQVVAIASHDGGKQALEAVRAQLPVLCQDHG Hybrid2 (SEQ ID NO: 336)LTPDQVVAIASHDGGKQALEAVWALLPVLCQDHG Hybrid3 (SEQ ID NO: 337)LTPDQVVAIASHDGGKPALEAVWAKLPVLCQDHG Hybrid4 (SEQ ID NO: 338)LSTAQVVAIASHDGGKQALETVQRLLPVLCQDHG Hybrid5 (SEQ ID NO: 339)LTPDQVVAIASHDGGKQALEAVRALFPDLCQDHG Hybrid6 (SEQ ID NO: 340)LTPDQVVAIASHDGGKQALETVQRLLPVLRQDHG Hybrid7 (SEQ ID NO: 341)LSTAQVVAIASHDGGKQALEAVRAQLPVLRGAHG Hybrid8

To explore this possibility, the repeat units shown below in Table 39were constructed. The table shows a typical Ralstonia repeat unit on thefirst line, and a Xanthomonas repeat unit on the second. Novel repeats,containing both Ralstonia derived residues and other variations designedto probe the sequence requirements for TALE repeats, are shown onsubsequent lines. All differences from the typical Xanthomonas repeatunit on the second line are underlined. Next, repeat units wereengineered by varying the positions that are in bold in rows 3-27. Thesenovel, engineered repeat units were then substituted into the systemdesigned to test the novel RVDs in Example 15 and shown in FIG. 27, andthe resultant constructs were translated in vitro and used in an ELISA.The target sequence used in the ELISA was the ‘C’ variant described inExample 15 (e.g. TTGACCATCC, SEQ ID NO:182) such that the RVD in all ofthese novel framework mutants was held constant at HD to interact withC. The ELISA results (average of 3 different experiments) are shown inTable 39 were all normalized to the standard sequence repeat unitsequence.

TABLE 39 Novel repeat framework substitutions Sequence ELISALSTAQVVAIASHDGGKQALEAVRAQLLVLRAAPYA ND (SEQ ID NO: 74)LTPDQVVAIASHDGGKQALETVQRLLPVLCQDHG 1.00 (SEQ ID NO: 333)LTPDAVVAIASHDGGKQALETVQRLLPVLCQDHG 1.03 (SEQ ID NO: 75)LTPDQAVAIASHDGGKQALETVQRLLPVLCQDHG 0.89 (SEQ ID NO: 76)LTPDQVAAIASHDGGKQALETVQRLLPVLCQDHG 0.26 (SEQ ID NO: 403)LTPDQVVLIASHDGGKQALETVQRLLPVLCQDHG 0.73 (SEQ ID NO: 404)LTPDQVVTIASHDGGKQALETVQRLLPVLCQDHG 0.82 (SEQ ID NO: 405)LTPDQVVAAASHDGGKQALETVQRLLPVLCQDHG 0.62 (SEQ ID NO: 406)LTPDQVVAVASHDGGKQALETVQRLLPVLCQDHG 0.76 (SEQ ID NO: 407)LTPDQVVAILSHDGGKQALETVQRLLPVLCQDHG 0.25 (SEQ ID NO: 408)LTPDQVVAIAAHDGGKQALETVQRLLPVLCQDHG 0.90 (SEQ ID NO: 409)LTPDQVVAIASHDGGKQALATVQRLLPVLCQDHG 0.82 (SEQ ID NO: 410)LTPDQVVAIASHDGGKQALEAVQRLLPVLCQDHG 1.05 (SEQ ID NO: 411)LTPDQVVAIASHDGGKQALETAQRLLPVLCQDHG 0.70 (SEQ ID NO: 412)LTPDQVVAIASHDGGKQALETVARLLPVLCQDHG 0.91 (SEQ ID NO: 413)LTPDQVVAIASHDGGKQALETVRRLLPVLCQDHG 0.97 (SEQ ID NO: 414)LTPDQVVAIASHDGGKQALETVKRLLPVLCQDHG 0.92 (SEQ ID NO: 415)LTPDQVVAIASHDGGKQALETVWRLLPVLCQDHG 0.88 (SEQ ID NO: 416)LTPDQVVAIASHDGGKQALETVQALLPVLCQDHG 0.92 (SEQ ID NO: 417)LTPDQVVAIASHDGGKQALETVQRALPVLCQDHG 1.09 (SEQ ID NO: 418)LTPDQVVAIASHDGGKQALETVQRQLPVLCQDHG 0.90 (SEQ ID NO: 419)LTPDQVVAIASHDGGKQALETVQRLAPVLCQDHG 1.00 (SEQ ID NO: 420)LTPDQVVAIASHDGGKQALETVQRLLAVLCQDHG 1.21 (SEQ ID NO: 421)LTPDQVVAIASHDGGKQALETVQRLLLVLCQDHG 1.29 (SEQ ID NO: 422)LTPDQVVAIASHDGGKQALEAVRALLPVLCQDHG 1.42 (SEQ ID NO: 423)LSTAQVVAIASHDGGKQALETVQRLLPVLCQDHG 0.00 (SEQ ID NO: 424)LSTAQVVAIASHDGGKQALEAVRALLPVLCQDHG 0.00 (SEQ ID NO: 425)

As can be seen from the ELISA results, the activity of TALE DNA bindingdomains comprising an engineered (e.g., novel) framework with mutationsin positions 2, 3, 4, 6, 7, 8, 9, 10 or 11 are diminished (withmutations in positions 2, 3, 4, 7, and 11 having the most significanteffect on binding). In contrast, many of the substitutions in positions20, 21, 24, 25, 26, and 27 either had a minimal effect on DNA binding oractually increased DNA binding. The largest increases in bindingoccurred when one or more residues from positions 21-27 in the Ralstoniarepeat were substituted into the Xanthomonas repeat.

Hybrid repeat units are combined in series to create novel TALE proteinsable to recognize any desired proteins. These novel TALE DNA bindingdomains are also linked to nuclease domains, transcriptional regulatorydomain, or any other active protein domain to cause a measurable resultfollowing DNA interaction.

Example 21 Construction of TALE-Zinc Finger DNA Binding Domain Hybrids

Zinc fingers were fused to TALE DNA binding domains to create a hybridDNA binding domain which was then linked to a nuclease. The target DNAsequences are shown below, and comprise a region surrounding a locuswithin the CCR5 gene. Shown above and below the binding site are thetarget binding sites for the TALE DNA binding domain and the zinc fingerbinding site is shown on the target sequence in Bold underline. The“TAG” sequence in Bold/underline is the binding site for the fourthfinger from the CCR5-specific ZFN SBS#8267, while the “AAACTG” sequencein Bold/underline is the binding site for the third and fourth fingersin the CCR5-specific ZFN SBS#8196 (see United States Patent Application11/805,707). The sequences below show that the zinc finger DNA targetsare not contiguous with the TALE DNA binding domain targets on the DNAstrand, creating the “inner gap”. Thus, this type of fusion allows thepractitioner to skip a region of DNA if desired within the inner gapregion.

  5′TTTGTGGGCAACAT (101025, SEQ ID NO: 455)   5′TTTGTGGGCAACATGCT(10126, SEQ ID NO: 456) 5′GTTTTGTGGGCAACATGCTGGTCATCCTCATCCTGAT AAACTGCAAAAGGCTGAAGAGCATGACTGACATCTAC   CAAAACACCCGTTGTACGACCAGTAGGAG TAGGACTATTTGACGTTTTCCGACTTCTCGTACTGACTGTAGATG 5′                            (101035, SEQ ID NO:457)        TTCCGACTTCTCGT 5′                               (101036, SEQID NO: 458)       TCCGACTTCTCGTACT 5′                                            (101037, SEQ ID NO:459)  TCTCGTACTGACTGT 5′                                            (101038, SEQ ID NO:460)    TCGTACTGACTGTAGAT

The table below, Table 40, shows the results of the studies. In thesestudies, one nuclease partner is field constant with an inner gap ofeither 7, 10, or 13 bases. The partner nuclease is then paired withproteins that comprise an inner gap of between 4 and 16 bases. As isshown in the table, TALE/zinc finger hybrid DNA binding domains can formactive nuclease pairs when the inner gaps range from 4 to 16 bases.

TABLE 40 Zinc Finger- TALE DNA binding domain hybrids Inner Inner gapgap (bp) (bp) 8267finger TALE- 8196finger TALE- Inter sample TALE- ZFPTALE- ZFP gap NHEJ # ZFN_L gap ZFN_R gap (bp) % 1 GFP <1.0 2 1-101025F413 6-101038F4 16 5 3.7 3 1-101025F4 13 7-101037F4 14 5 6.8 4 1-101025F413 8-101036F4 7 5 11.7 5 1-101025F4 13 9-101035F4 6 5 16.1 6 1-101025F413 10- 13 5 26.4 101038F34 7 1-101025F4 13 11- 11 5 13.4 101037F34 81-101025F4 13 12- 4 5 1.9 101036F34 9 101028 101036 24.5 10 2-101025F3410 6-101038F4 16 5 23.6 11 2-101025F34 10 7-101037F4 14 5 14.4 122-101025F34 10 8-101036F4 7 5 12.4 13 2-101025F34 10 9-101035F4 6 5 18.114 2- 10 10- 13 5 32.2 101025F34 101038F34 15 2- 10 11- 11 5 31.4101025F34 101037F34 16 2- 10 12- 4 5 8.1 101025F34 101036F34 17 82678196 49.6 18 3-101026F4 10 6-101038F4 16 5 <1.0 19 3-101026F4 107-101037F4 14 5 <1.0 20 3-101026F4 10 8-101036F4 7 5 <1.0 21 3-101026F410 9-101035F4 6 5 <1.0 22 3-101026F4 10 10- 13 5 6.4 101038F34 233-101026F4 10 11- 11 5 10.7 101037F34 24 3-101026F4 10 12- 4 5 <1.0101036F34 25 8267 101036 1.8 26 4- 7 6-101038F4 16 5 34.1 101026F34 274- 7 7-101037F4 14 5 17.3 101026F34 28 4- 7 8-101036F4 7 5 12.6101026F34 29 4- 7 9-101035F4 6 5 53.3 101026F34 30 4- 7 10- 13 5 42.6101026F34 101038F34 31 4- 7 11- 11 5 44.7 101026F34 101037F34 32 4- 712- 4 5 36.3 101026F34 101036F34

Example 22 Construction of a TALE-Integrase Fusion Protein

During the life cycle of retroviruses, viral genomic RNAs are reversetranscribed and integrated at many different sites into host genome,even though there are preferences for certain hot spots. Forapplications utilizing retroviral vectors, especially gene therapy, thepossible carcinogenicity of retroviral vectors due to random integrationof engineered viral genome near oncogene loci presents a potential riskfactor. To overcome such potential problems, the specificity of viralintegrases is re-directed to pre-determined sites by utilizing specificTALE DNA-binding domains. Fusions are made with whole or truncatedintegrases and with whole or truncated integrase-binding proteins (forexample LEDGF for HIV integrase).

Additionally, fusion pairs are made where one member of the pair is anintegrate fused to one protein (for example protein1) and the secondpair is a fusion of a TALE DNA binding domain with another protein (forexample protein2) where protein1 and protein2 bind to each other. Thefusion pairs are cloned into an expression vector such that the pair isexpressed in the cell of interest. For a mammalian genomic target, thefusion pair is expressed using a mammalian expression vector. Duringexpression of the TALEN fusions, a donor DNA is supplied such that thedonor is incorporated into the cleavage site following TALEN-induced DNAfusion.

Example 23 Sequences of Various TALE Constructs DNA and ProteinSequences

Complete TALEN Construct Sequence, with Coding Sequence Underlined (SEQID NO:217):

GACTCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTGATCCACTAGTCCAGTGTGGTGGAATTCGCCATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACGGGGTACCCGCCGCTGTGGATCTACGCACGCTCGGCTACAGCCAGCAGCAACAGGAGAAGATCAAACCGAAGGTTCGTTCGACAGTGGCGCAGCACCACGAGGCACTGGTCGGCCATGGGTTTACACACGCGCACATCGTTGCGCTCAGCCAACACCCGGCAGCGTTAGGGACCGTCGCTGTCAAGTATCAGGACATGATCGCAGCGTTGCCAGAGGCGACACACGAAGCGATCGTTGGCGTCGGCAAACAGTGGTCCGGCGCACGCGCCCTGGAGGCCTTGCTCACGGTGGCGGGAGAGTTGAGAGGTCCACCGTTACAGTTGGACACAGGCCAACTTCTCAAGATTGCAAAACGTGGCGGCGTGACCGCAGTGGAGGCAGTGCATGCATGGCGCAATGCACTGACGGGGGCCCCCCTGAACCTTACTCCCGAACAAGTAGTAGCGATAGCCAGTAATAACGGAGGTAAACAAGCCTTGGAGACGGTCCAAAGGTTGCTCCCGGTCTTGTGTCAGGCACATGGGCTGACGCCTCAACAGGTCGTCGCGATAGCGTCTAATAATGGAGGAAAGCAAGCTCTGGAAACCGTCCAGCGACTCCTTCCGGTTCTGTGCCAGGCTCATGGTCTGACTCCGCAGCAAGTCGTTGCTATAGCGTCCAACATCGGAGGCAAACAGGCCCTGGAGACCGTGCAGCGGTTGTTGCCTGTGCTTTGCCAAGCCCACGGGCTTACGCCTGAGCAAGTGGTGGCGATTGCCAGTAACAACGGCGGCAAACAAGCCCTTGAGACTGTGCAGAGGCTCTTGCCGGTACTCTGCCAAGCACACGGCTTGACCCCCGAGCAGGTTGTAGCCATAGCTAGTCACGACGGGGGTAAGCAAGCGTTGGAAACGGTGCAAGCACTTCTCCCCGTTCTCTGTCAAGCGCATGGACTTACCCCGGAACAGGTGGTCGCCATTGCAAGCCATGATGGAGGAAAGCAGGCGCTCGAAACAGTCCAGGCACTTTTGCCCGTACTTTGTCAAGCTCACGGTCTCACCCCGGAACAGGTGGTAGCCATTGCATCTAACATCGGAGGTAAGCAAGCATTGGAAACGGTTCAGGCCCTGTTGCCTGTACTTTGCCAGGCGCACGGTCTGACACCTGAGCAGGTTGTCGCCATCGCTAGCAACGGAGGTGGGAAACAGGCACTTGAAACTGTGCAGAGGCTTCTGCCGGTGCTGTGCCAAGCGCATGGCCTTACACCCGAGCAAGTAGTGGCTATTGCGAGTCATGATGGAGGCAAGCAAGCGCTGGAGACTGTCCAACGACTTCTTCCGGTCTTGTGTCAGGCACATGGATTGACCCCTCAACAAGTCGTGGCGATAGCTAGCAACGGCGGTGGAAAACAGGCCCTCGAAACCGTCCAGCGACTGCTCCCCGTACTGTGTCAAGCCCATGGACTTACCCCAGAACAAGTTGTGGCGATTGCCTCTAACAATGGTGGGAAGCAAGCTCTTGAGACGGTGCAGGCGTTGTTGCCCGTGCTTTGTCAAGCTCACGGGCTCACGCCAGAGCAAGTGGTCGCTATCGCGAGTAATAAAGGGGGCAAACAAGCCTTGGAGACAGTGCAAAGGCTCCTGCCAGTGCTCTGCCAGGCTCATGGTTTGACACCCGAACAGGTAGTTGCAATAGCGAGTCATGATGGCGGAAAGCAAGCTCTTGAAACTGTGCAGCGGCTGTTGCCTGTACTGTGTCAAGCCCACGGGCTGACACCGGAACAAGTTGTAGCGATCGCTAGCCACGATGGCGGGAAACAAGCTCTGGAAACGGTACAGAGACTCCTCCCAGTGCTTTGTCAGGCACACGGCCTCACGCCAGAGCAGGTTGTCGCCATCGCGTCAAACAATGGTGGAAAGCAGGCCCTGGAGACAGTCCAACGGTTGCTGCCGGTCCTTTGCCAGGCTCACGGGTTGACCCCCCAGCAGGTCGTGGCCATTGCCTCAAACAAGGGCGGTAGGCCAGCATTGGAGACGGTGCAGAGGCTTCTGCCTGTGCTCTGCCAAGCGCATGGACTCACCCCCGAGCAAGTGGTTGCTATCGCAAGTAACAACGGAGGGAAACAAGCGCTCGAAACCGTGCAAAGGTTGCTCCCCGTTCTCTGTCAGGCGCACGGTCTTACGCCACAACAGGTGGTGGCGATTGCATCTAATGGAGGCGGACGCCCTGCCTTGGAGAGCATTGTGGCCCAGCTGTCCAGGCCGGACCCTGCCCTGGCCGCGTTAACCAACGACCACCTCGTCGCCTTGGCCTGCCTCGGCGGAGGTTCTGGCGGCAGCGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCTTGATAACTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAATTGCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGGATGGCTTTCTCGCCGCCAAGGATCTGATGGCGCAGGGGATCAAGCTCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGAGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAATTATTAACGCTTACAATTTCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATAGCACGTGCTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGGCTTTTGCTGGCCTTTTGCTCACATGTTCTT

Complete Protein and Coding Sequence for Each TALEN Used in NTF3Modification and In Vitro Cleavage Studies

To regenerate the sequence of each expression construct, replace theunderlined region of the above construct with each CDS shown below.

>NT_L +28 (SEQ ID NO: 218)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAAVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPEQVVAIASNKGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNKGGRPALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGGSGGSGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS >NT_L +28 (SEQ ID NO: 219)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACGGGGTACCCGCCGCTGTGGATCTACGCACGCTCGGCTACAGCCAGCAGCAACAGGAGAAGATCAAACCGAAGGTTCGTTCGACAGTGGCGCAGCACCACGAGGCACTGGTCGGCCATGGGTTTACACACGCGCACATCGTTGCGCTCAGCCAACACCCGGCAGCGTTAGGGACCGTCGCTGTCAAGTATCAGGACATGATCGCAGCGTTGCCAGAGGCGACACACGAAGCGATCGTTGGCGTCGGCAAACAGTGGTCCGGCGCACGCGCCCTGGAGGCCTTGCTCACGGTGGCGGGAGAGTTGAGAGGTCCACCGTTACAGTTGGACACAGGCCAACTTCTCAAGATTGCAAAACGTGGCGGCGTGACCGCAGTGGAGGCAGTGCATGCATGGCGCAATGCACTGACGGGGGCCCCCCTGAACCTTACTCCCGAACAAGTAGTAGCGATAGCCAGTAATAACGGAGGTAAACAAGCCTTGGAGACGGTCCAAAGGTTGCTCCCGGTCTTGTGTCAGGCACATGGGCTGACGCCTCAACAGGTCGTCGCGATAGCGTCTAATAATGGAGGAAAGCAAGCTCTGGAAACCGTCCAGCGACTCCTTCCGGTTCTGTGCCAGGCTCATGGTCTGACTCCGCAGCAAGTCGTTGCTATAGCGTCCAACATCGGAGGCAAACAGGCCCTGGAGACCGTGCAGCGGTTGTTGCCTGTGCTTTGCCAAGCCCACGGGCTTACGCCTGAGCAAGTGGTGGCGATTGCCAGTAACAACGGCGGCAAACAAGCCCTTGAGACTGTGCAGAGGCTCTTGCCGGTACTCTGCCAAGCACACGGCTTGACCCCCGAGCAGGTTGTAGCCATAGCTAGTCACGACGGGGGTAAGCAAGCGTTGGAAACGGTGCAAGCACTTCTCCCCGTTCTCTGTCAAGCGCATGGACTTACCCCGGAACAGGTGGTCGCCATTGCAAGCCATGATGGAGGAAAGCAGGCGCTCGAAACAGTCCAGGCACTTTTGCCCGTACTTTGTCAAGCTCACGGTCTCACCCCGGAACAGGTGGTAGCCATTGCATCTAACATCGGAGGTAAGCAAGCATTGGAAACGGTTCAGGCCCTGTTGCCTGTACTTTGCCAGGCGCACGGTCTGACACCTGAGCAGGTTGTCGCCATCGCTAGCAACGGAGGTGGGAAACAGGCACTTGAAACTGTGCAGAGGCTTCTGCCGGTGCTGTGCCAAGCGCATGGCCTTACACCCGAGCAAGTAGTGGCTATTGCGAGTCATGATGGAGGCAAGCAAGCGCTGGAGACTGTCCAACGACTTCTTCCGGTCTTGTGTCAGGCACATGGATTGACCCCTCAACAAGTCGTGGCGATAGCTAGCAACGGCGGTGGAAAACAGGCCCTCGAAACCGTCCAGCGACTGCTCCCCGTACTGTGTCAAGCCCATGGACTTACCCCAGAACAAGTTGTGGCGATTGCCTCTAACAATGGTGGGAAGCAAGCTCTTGAGACGGTGCAGGCGTTGTTGCCCGTGCTTTGTCAAGCTCACGGGCTCACGCCAGAGCAAGTGGTCGCTATCGCGAGTAATAAAGGGGGCAAACAAGCCTTGGAGACAGTGCAAAGGCTCCTGCCAGTGCTCTGCCAGGCTCATGGTTTGACACCCGAACAGGTAGTTGCAATAGCGAGTCATGATGGCGGAAAGCAAGCTCTTGAAACTGTGCAGCGGCTGTTGCCTGTACTGTGTCAAGCCCACGGGCTGACACCGGAACAAGTTGTAGCGATCGCTAGCCACGATGGCGGGAAACAAGCTCTGGAAACGGTACAGAGACTCCTCCCAGTGCTTTGTCAGGCACACGGCCTCACGCCAGAGCAGGTTGTCGCCATCGCGTCAAACAATGGTGGAAAGCAGGCCCTGGAGACAGTCCAACGGTTGCTGCCGGTCCTTTGCCAGGCTCACGGGTTGACCCCCCAGCAGGTCGTGGCCATTGCCTCAAACAAGGGCGGTAGGCCAGCATTGGAGACGGTGCAGAGGCTTCTGCCTGTGCTCTGCCAAGCGCATGGACTCACCCCCGAGCAAGTGGTTGCTATCGCAAGTAACAACGGAGGGAAACAAGCGCTCGAAACCGTGCAAAGGTTGCTCCCCGTTCTCTGTCAGGCGCACGGTCTTACGCCACAACAGGTGGTGGCGATTGCATCTAATGGAGGCGGACGCCCTGCCTTGGAGAGCATTGTGGCCCAGCTGTCCAGGCCGGACCCTGCCCTGGCCGCGTTAACCAACGACCACCTCGTCGCCTTGGCCTGCCTCGGCGGAGGTTCTGGCGGCAGCGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT >NT_L +63 (SEQ ID NO: 220)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPEQVVAIASNKGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNKGGRPALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS >NT_L+63 (SEQ ID NO: 221)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACGGGGTACCCATGGTGGATCTACGCACGCTCGGCTACAGCCAGCAGCAACAGGAGAAGATCAAACCGAAGGTTCGTTCGACAGTGGCGCAGCACCACGAGGCACTGGTCGGCCATGGGTTTACACACGCGCACATCGTTGCGCTCAGCCAACACCCGGCAGCGTTAGGGACCGTCGCTGTCAAGTATCAGGACATGATCGCAGCGTTGCCAGAGGCGACACACGAAGCGATCGTTGGCGTCGGCAAACAGTGGTCCGGCGCACGCGCCCTGGAGGCCTTGCTCACGGTGGCGGGAGAGTTGAGAGGTCCACCGTTACAGTTGGACACAGGCCAACTTCTCAAGATTGCAAAACGTGGCGGCGTGACCGCAGTGGAGGCAGTGCATGCATGGCGCAATGCACTGACGGGGGCCCCCCTGAACCTTACTCCCGAACAAGTAGTAGCGATAGCCAGTAATAACGGAGGTAAACAAGCCTTGGAGACGGTCCAAAGGTTGCTCCCGGTCTTGTGTCAGGCACATGGGCTGACGCCTCAACAGGTCGTCGCGATAGCGTCTAATAATGGAGGAAAGCAAGCTCTGGAAACCGTCCAGCGACTCCTTCCGGTTCTGTGCCAGGCTCATGGTCTGACTCCGCAGCAAGTCGTTGCTATAGCGTCCAACATCGGAGGCAAACAGGCCCTGGAGACCGTGCAGCGGTTGTTGCCTGTGCTTTGCCAAGCCCACGGGCTTACGCCTGAGCAAGTGGTGGCGATTGCCAGTAACAACGGCGGCAAACAAGCCCTTGAGACTGTGCAGAGGCTCTTGCCGGTACTCTGCCAAGCACACGGCTTGACCCCCGAGCAGGTTGTAGCCATAGCTAGTCACGACGGGGGTAAGCAAGCGTTGGAAACGGTGCAAGCACTTCTCCCCGTTCTCTGTCAAGCGCATGGACTTACCCCGGAACAGGTGGTCGCCATTGCAAGCCATGATGGAGGAAAGCAGGCGCTCGAAACAGTCCAGGCACTTTTGCCCGTACTTTGTCAAGCTCACGGTCTCACCCCGGAACAGGTGGTAGCCATTGCATCTAACATCGGAGGTAAGCAAGCATTGGAAACGGTTCAGGCCCTGTTGCCTGTACTTTGCCAGGCGCACGGTCTGACACCTGAGCAGGTTGTCGCCATCGCTAGCAACGGAGGTGGGAAACAGGCACTTGAAACTGTGCAGAGGCTTCTGCCGGTGCTGTGCCAAGCGCATGGCCTTACACCCGAGCAAGTAGTGGCTATTGCGAGTCATGATGGAGGCAAGCAAGCGCTGGAGACTGTCCAACGACTTCTTCCGGTCTTGTGTCAGGCACATGGATTGACCCCTCAACAAGTCGTGGCGATAGCTAGCAACGGCGGTGGAAAACAGGCCCTCGAAACCGTCCAGCGACTGCTCCCCGTACTGTGTCAAGCCCATGGACTTACCCCAGAACAAGTTGTGGCGATTGCCTCTAACAATGGTGGGAAGCAAGCTCTTGAGACGGTGCAGGCGTTGTTGCCCGTGCTTTGTCAAGCTCACGGGCTCACGCCAGAGCAAGTGGTCGCTATCGCGAGTAATAAAGGGGGCAAACAAGCCTTGGAGACAGTGCAAAGGCTCCTGCCAGTGCTCTGCCAGGCTCATGGTTTGACACCCGAACAGGTAGTTGCAATAGCGAGTCATGATGGCGGAAAGCAAGCTCTTGAAACTGTGCAGCGGCTGTTGCCTGTACTGTGTCAAGCCCACGGGCTGACACCGGAACAAGTTGTAGCGATCGCTAGCCACGATGGCGGGAAACAAGCTCTGGAAACGGTACAGAGACTCCTCCCAGTGCTTTGTCAGGCACACGGCCTCACGCCAGAGCAGGTTGTCGCCATCGCGTCAAACAATGGTGGAAAGCAGGCCCTGGAGACAGTCCAACGGTTGCTGCCGGTCCTTTGCCAGGCTCACGGGTTGACCCCCCAGCAGGTCGTGGCCATTGCCTCAAACAAGGGCGGTAGGCCAGCATTGGAGACGGTGCAGAGGCTTCTGCCTGTGCTCTGCCAAGCGCATGGACTCACCCCCGAGCAAGTGGTTGCTATCGCAAGTAACAACGGAGGGAAACAAGCGCTCGAAACCGTGCAAAGGTTGCTCCCCGTTCTCTGTCAGGCGCACGGTCTTACGCCACAACAGGTGGTGGCGATTGCATCTAATGGAGGCGGACGCCCTGCCTTGGAGAGCATTGTGGCCCAGCTGTCCAGGCCGGACCCTGCCCTGGCCGCGTTAACCAACGACCACCTCGTCGCCTTGGCCTGCCTCGGCGGACGTCCTGCGCTGGATGCAGTGAAAAAGGGATTGCCGCACGCGCCGGCCTTGATCAAAAGAACCAATCGCCGTATTCCCGAACGCACATCCCATCGCGTTGCCGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT >NT_R +28 (SEQ ID NO: 222)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKAGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNKGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNKGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNKGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS >NT_R +28 (SEQ ID NO: 223)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACGGGGTACCTATGGTGGACTTGAGGACACTCGGTTATTCGCAACAGCAACAGGAGAAAATCAAGCCTAAGGTCAGGAGCACCGTCGCGCAACACCACGAGGCGCTTGTGGGGCATGGCTTCACTCATGCGCATATTGTCGCGCTTTCACAGCACCCTGCGGCGCTTGGGACGGTGGCTGTCAAATACCAAGATATGATTGCGGCCCTGCCCGAAGCCACGCACGAGGCAATTGTAGGGGTCGGTAAACAGTGGTCGGGAGCGCGAGCACTTGAGGCGCTGCTGACTGTGGCGGGTGAGCTTAGGGGGCCTCCGCTCCAGCTCGACACCGGGCAGCTGCTGAAGATCGCGAAGAGAGGGGGAGTAACAGCGGTAGAGGCAGTGCACGCCTGGCGCAATGCGCTCACCGGGGCCCCCTTGAATCTTACTCCAGAGCAGGTCGTCGCAATCGCGTCGAATAACGGGGGAAAGCAAGCACTGGAAACCGTGCAGAGGTTGTTGCCGGTCTTGTGTCAGGCTCACGGCTTGACACCTGCCCAAGTGGTGGCCATTGCGTCGAACATCGGGGGAAAACAGGCACTTGAAACAGTCCAGAGACTTTTGCCCGTCCTCTGCCAGGCGCACGGCCTCACGCCGGATCAGGTGGTAGCCATCGCGTCAAACATCGGAGGGAAGCAGGCTCTGGAAACGGTGCAGCGGCTTTTGCCGGTACTTTGCCAAGCTCATGGGCTCACGCCAGCCCAAGTGGTAGCTATCGCATCGCACGACGGAGGGAAGCAGGCCTTGGAGACAGTGCAACGGCTCCTCCCCGTGTTGTGCCAGGCACATGGGTTGACTCCAGAGCAGGTCGTAGCAATCGCCTCCAATATCGGGGGAAAGCAAGCGTTGGAGACAGTGCAGCGACTGCTGCCTGTGCTTTGCCAGGCTCATGGCCTGACGCCCGATCAGGTAGTGGCAATCGCGTCAAACAAAGGTGGAAAGCAGGCACTCGAAACGGTACAGCGCTTGCTGCCCGTCTTGTGTCAGGCCCACGGTCTGACACCCGACCAGGTAGTCGCGATTGCGTCGAACATCGGGGGAAAGCAAGCGTTGGAAACGGTACAACGCCTGCTCCCGGTGCTCTGCCAGGCTCATGGACTTACACCCGAGCAGGTGGTCGCCATCGCGTCAAACATCGGAGGCAAACAGGCATTGGAGACAGTGCAGCGCCTTCTCCCAGTCTTGTGTCAGGCCCACGGTCTGACACCCGACCAGGTCGTCGCGATTGCATCGAATGGAGGTGGGAAACAGGCCCTTGAGACAGTACAGAGGCTTTTGCCCGTGTTGTGCCAGGCCCACGGACTCACACCCGAACAAGTCGTCGCCATTGCCAGCCATGATGGAGGTAAACAGGCACTTGAGACTGTCCAGCGCCTCCTGCCGGTGCTGTGCCAAGCACATGGGCTGACCCCGCAGCAAGTCGTAGCGATCGCCTCGAATGGTGGAGGAAAACAAGCGCTTGAAACCGTCCAGAGGTTGCTCCCGGTGCTGTGCCAGGCACATGGCCTTACGCCTGAACAAGTAGTCGCGATTGCCAGCAACAAAGGCGGAAAACAGGCTCTCGAAACGGTCCAGCGGTTGCTGCCGGTGTTGTGCCAGGCGCACGGTCTTACACCGGACCAGGTGGTGGCGATTGCCTCCCACGATGGGGGTAAACAGGCACTGGAAACCGTGCAGAGATTGCTCCCAGTACTTTGTCAGGCACATGGTCTGACTCCTGCTCAAGTGGTCGCGATCGCCTCGAACAATGGCGGAAAGCAGGCGCTCGAAACGGTACAGCGGCTCCTTCCGGTGCTCTGCCAAGCCCACGGATTGACGCCAGAACAGGTCGTGGCAATTGCGTCACACGACGGTGGAAAGCAGGCGCTCGAAACTGTGCAAAGACTCCTGCCCGTACTCTGCCAGGCACACGGTTTGACTCCCCAGCAGGTAGTGGCCATCGCGAGCAATAAGGGAGGAAAGCAGGCGCTTGAAACGGTGCAGAGACTTCTGCCCGTGCTTTGTCAAGCCCACGGGCTGACTCCGGAGCAGGTAGTGGCCATCGCCTCAAACAACGGAGGAAAGCAAGCTCTCGAAACCGTACAGAGGCTTCTCCCCGTGCTCTGTCAGGCCCACGGGTTGACCCCCGAACAGGTGGTCGCCATTGCTAGCAACGGGGGAGGACGGCCAGCCTTGGAGTCCATCGTAGCCCAATTGTCCAGGCCCGATCCCGCGTTGGCTGCGTTAACGAATGACCATCTGGTGGCGTTGGCATGTCTTGGTGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT >NT_R+63, (also referred to as rNT3 C+63) (SEQ ID NO: 224)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNKGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNKGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNKGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS >NT_R+63 (SEQ ID NO: 225)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACGGGGTACCTATGGTGGACTTGAGGACACTCGGTTATTCGCAACAGCAACAGGAGAAAATCAAGCCTAAGGTCAGGAGCACCGTCGCGCAACACCACGAGGCGCTTGTGGGGCATGGCTTCACTCATGCGCATATTGTCGCGCTTTCACAGCACCCTGCGGCGCTTGGGACGGTGGCTGTCAAATACCAAGATATGATTGCGGCCCTGCCCGAAGCCACGCACGAGGCAATTGTAGGGGTCGGTAAACAGTGGTCGGGAGCGCGAGCACTTGAGGCGCTGCTGACTGTGGCGGGTGAGCTTAGGGGGCCTCCGCTCCAGCTCGACACCGGGCAGCTGCTGAAGATCGCGAAGAGAGGGGGAGTAACAGCGGTAGAGGCAGTGCACGCCTGGCGCAATGCGCTCACCGGGGCCCCCTTGAATCTTACTCCAGAGCAGGTCGTCGCAATCGCGTCGAATAACGGGGGAAAGCAAGCACTGGAAACCGTGCAGAGGTTGTTGCCGGTCTTGTGTCAGGCTCACGGCTTGACACCTGCCCAAGTGGTGGCCATTGCGTCGAACATCGGGGGAAAACAGGCACTTGAAACAGTCCAGAGACTTTTGCCCGTCCTCTGCCAGGCGCACGGCCTCACGCCGGATCAGGTGGTAGCCATCGCGTCAAACATCGGAGGGAAGCAGGCTCTGGAAACGGTGCAGCGGCTTTTGCCGGTACTTTGCCAAGCTCATGGGCTCACGCCAGCCCAAGTGGTAGCTATCGCATCGCACGACGGAGGGAAGCAGGCCTTGGAGACAGTGCAACGGCTCCTCCCCGTGTTGTGCCAGGCACATGGGTTGACTCCAGAGCAGGTCGTAGCAATCGCCTCCAATATCGGGGGAAAGCAAGCGTTGGAGACAGTGCAGCGACTGCTGCCTGTGCTTTGCCAGGCTCATGGCCTGACGCCCGATCAGGTAGTGGCAATCGCGTCAAACAAAGGTGGAAAGCAGGCACTCGAAACGGTACAGCGCTTGCTGCCCGTCTTGTGTCAGGCCCACGGTCTGACACCCGACCAGGTAGTCGCGATTGCGTCGAACATCGGGGGAAAGCAAGCGTTGGAAACGGTACAACGCCTGCTCCCGGTGCTCTGCCAGGCTCATGGACTTACACCCGAGCAGGTGGTCGCCATCGCGTCAAACATCGGAGGCAAACAGGCATTGGAGACAGTGCAGCGCCTTCTCCCAGTCTTGTGTCAGGCCCACGGTCTGACACCCGACCAGGTCGTCGCGATTGCATCGAATGGAGGTGGGAAACAGGCCCTTGAGACAGTACAGAGGCTTTTGCCCGTGTTGTGCCAGGCCCACGGACTCACACCCGAACAAGTCGTCGCCATTGCCAGCCATGATGGAGGTAAACAGGCACTTGAGACTGTCCAGCGCCTCCTGCCGGTGCTGTGCCAAGCACATGGGCTGACCCCGCAGCAAGTCGTAGCGATCGCCTCGAATGGTGGAGGAAAACAAGCGCTTGAAACCGTCCAGAGGTTGCTCCCGGTGCTGTGCCAGGCACATGGCCTTACGCCTGAACAAGTAGTCGQGATTGCCAGCAACAAAGGCGGAAAACAGGCTCTCGAAACGGTCCAGCGGTTGCTGCCGGTGTTGTGCCAGGCGCACGGTCTTACACCGGACCAGGTGGTGGCGATTGCCTCCCACGATGGGGGTAAACAGGCACTGGAAACCGTGCAGAGATTGCTCCCAGTACTTTGTCAGGCACATGGTCTGACTCCTGCTCAAGTGGTCGCGATCGCCTCGAACAATGGCGGAAAGCAGGCGCTCGAAACGGTACAGCGGCTCCTTCCGGTGCTCTGCCAAGCCCACGGATTGACGCCAGAACAGGTCGTGGCAATTGCGTCACACGACGGTGGAAAGCAGGCGCTCGAAACTGTGCAAAGACTCCTGCCCGTACTCTGCCAGGCACACGGTTTGACTCCCCAGCAGGTAGTGGCCATCGCGAGCAATAAGGGAGGAAAGCAGGCGCTTGAAACGGTGCAGAGACTTCTGCCCGTGCTTTGTCAAGCCCACGGGCTGACTCCGGAGCAGGTAGTGGCCATCGCCTCAAACAACGGAGGAAAGCAAGCTCTCGAAACCGTACAGAGGCTTCTCCCCGTGCTCTGTCAGGCCCACGGGTTGACCCCCGAACAGGTGGTCGCCATTGCTAGCAACGGGGGAGGACGGCCAGCCTTGGAGTCCATCGTAGCCCAATTGTCCAGGCCCGATCCCGCGTTGGCTGCGTTAACGAATGACCATCTGGTGGCGTTGGCATGTCTTGGTGGACGACCCGCGCTCGATGCAGTCAAAAAGGGTCTGCCTCATGCTCCCGCATTGATCAAAAGAACCAACCGGCGGATTCCCGAGAGAACTTCCCATCGAGTCGCGGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT >TALE13 +28 (also referred to as rNT#C+28) (SEQ ID NO: 226)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLRQAHGLTPEQVVAIASNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPSLAALTNDHLVALACLGGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS >TALE13_+28 (SEQID NO: 227)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACGGGGTACCCATGGTGGATCTACGCACGCTCGGCTACAGCCAGCAGCAACAGGAGAAGATCAAACCGAAGGTTCGTTCGACAGTGGCGCAGCACCACGAGGCACTGGTCGGCCATGGGTTTACACACGCGCACATCGTTGCGCTCAGCCAACACCCGGCAGCGTTAGGGACCGTCGCTGTCAAGTATCAGGACATGATCGCAGCGTTGCCAGAGGCGACACACGAAGCGATCGTTGGCGTCGGCAAACAGTGGTCCGGCGCACGCGCCCTGGAGGCCTTGCTCACGGTGGCGGGAGAGTTGAGAGGTCCACCGTTACAGTTGGACACAGGCCAACTTCTCAAGATTGCAAAACGTGGCGGCGTGACCGCAGTGGAGGCAGTGCATGCATGGCGCAATGCACTGACGGGGGCCCCCCTGAACCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGCACAGGTGGTGGCCATCGCCAGCAATATTGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGCGCCAGGCCCATGGCCTGACCCCGGAGCAGGTCGTGGCCATCGCCAGCAATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGCACAGGTGGTGGCCATCGCCAGCAATATTGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTCGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGACCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGACCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATGGCGGCGGCAGGCCGGCGCTGGAGAGCATTGTTGCCCAGTTATCTCGCCCTGATCCGTCGTTGGCCGCGTTAACCAACGACCACCTCGTCGCCTTGGCCTGCCTCGGCGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT >TALE13+39, (also referred to as rNT3, C+39) (SEQ ID NO: 228)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIARAGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLRQAHGLTPEQVVAIASNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPSLAALTNDHLVALACLGGRPALDAVKKGGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS >TALE13+39 (SEQ ID NO: 229)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACGGGGTACCCATGGTGGATCTACGCACGCTCGGCTACAGCCAGCAGCAACAGGAGAAGATCAAACCGAAGGTTCGTTCGACAGTGGCGCAGCACCACGAGGCACTGGTCGGCCATGGGTTTACACACGCGCACATCGTTGCGCTCAGCCAACACCCGGCAGCGTTAGGGACCGTCGCTGTCAAGTATCAGGACATGATCGCAGCGTTGCCAGAGGCGACACACGAAGCGATCGTTGGCGTCGGCAAACAGTGGTCCGGCGCACGCGCCCTGGAGGCCTTGCTCACGGTGGCGGGAGAGTTGAGAGGTCCACCGTTACAGTTGGACACAGGCCAACTTCTCAAGATTGCAAAACGTGGCGGCGTGACCGCAGTGGAGGCAGTGCATGCATGGCGCAATGCACTGACGGGGGCCCCCCTGAACCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGCACAGGTGGTGGCCATCGCCAGCAATATTGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGCGCCAGGCCCATGGCCTGACCCCGGAGCAGGTCGTGGCCATCGCCAGCAATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGCACAGGTGGTGGCCATCGCCAGCAATATTGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTCGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGACCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGACCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATGGCGGCGGCAGGCCGGCGCTGGAGAGCATTGTTGCCCAGTTATCTCGCCCTGATCCGTCGTTGGCCGCGTTAACCAACGACCACCTCGTCGCCTTGGCCTGCCTCGGCGGACGTCCTGCGCTGGATGCAGTGAAAAAGGGAGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT >TALE13+50 (SEQ ID NO: 230)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLRQAHGLTPEQVVAIASNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPSLAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS >TALE13+50 (SEQ ID NO: 231)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACGGGGTACCCATGGTGGATCTACGCACGCTCGGCTACAGCCAGCAGCAACAGGAGAAGATCAAACCGAAGGTTCGTTCGACAGTGGCGCAGCACCACGAGGCACTGGTCGGCCATGGGTTTACACACGCGCACATCGTTGCGCTCAGCCAACACCCGGCAGCGTTAGGGACCGTCGCTGTCAAGTATCAGGACATGATCGCAGCGTTGCCAGAGGCGACACACGAAGCGATCGTTGGCGTCGGCAAACAGTGGTCCGGCGCACGCGCCCTGGAGGCCTTGCTCACGGTGGCGGGAGAGTTGAGAGGTCCACCGTTACAGTTGGACACAGGCCAACTTCTCAAGATTGCAAAACGTGGCGGCGTGACCGCAGTGGAGGCAGTGCATGCATGGCGCAATGCACTGACGGGGGCCCCCCTGAACCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGCACAGGTGGTGGCCATCGCCAGCAATATTGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGCGCCAGGCCCATGGCCTGACCCCGGAGCAGGTCGTGGCCATCGCCAGCAATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGCACAGGTGGTGGCCATCGCCAGCAATATTGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTCGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGACCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGACCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATGGCGGCGGCAGGCCGGCGCTGGAGAGCATTGTTGCCCAGTTATCTCGCCCTGATCCGTCGTTGGCCGCGTTAACCAACGACCACCTCGTCGCCTTGGCCTGCCTCGGCGGACGTCCTGCGCTGGATGCAGTGAAAAAGGGATTGCCGCACGCGCCGGCCTTGATCAAAAGAACCGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT >TALE13 +63 (SEQ ID NO: 232)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEATVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLRQAHGLTPEQVVAIASNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPSLAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS >TALE13+63 (SEQ ID NO: 233)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACGGGGTACCCATGGTGGATCTACGCACGCTCGGCTACAGCCAGCAGCAACAGGAGAAGATCAAACCGAAGGTTCGTTCGACAGTGGCGCAGCACCACGAGGCACTGGTCGGCCATGGGTTTACACACGCGCACATCGTTGCGCTCAGCCAACACCCGGCAGCGTTAGGGACCGTCGCTGTCAAGTATCAGGACATGATCGCAGCGTTGCCAGAGGCGACACACGAAGCGATCGTTGGCGTCGGCAAACAGTGGTCCGGCGCACGCGCCCTGGAGGCCTTGCTCACGGTGGCGGGAGAGTTGAGAGGTCCACCGTTACAGTTGGACACAGGCCAACTTCTCAAGATTGCAAAACGTGGCGGCGTGACCGCAGTGGAGGCAGTGCATGCATGGCGCAATGCACTGACGGGGGCCCCCCTGAACCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGCACAGGTGGTGGCCATCGCCAGCAATATTGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGCGCCAGGCCCATGGCCTGACCCCGGAGCAGGTCGTGGCCATCGCCAGCAATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGCACAGGTGGTGGCCATCGCCAGCAATATTGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTCGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGACCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGACCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATGGCGGCGGCAGGCCGGCGCTGGAGAGCATTGTTGCCCAGTTATCTCGCCCTGATCCGTCGTTGGCCGCGTTAACCAACGACCACCTCGTCGCCTTGGCCTGCCTCGGCGGACGTCCTGCGCTGGATGCAGTGAAAAAGGGATTGCCGCACGCGCCGGCCTTGATCAAAAGAACCAATCGCCGTATTCCCGAACGCACATCCCATCGCGTTGCCGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT >TALE13+79 (SEQ ID NO: 234)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLAMTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLRQAHGLTPEQVVAIASNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPSLAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVARKFNNGEINFRS >TALE13 +79 (SEQ ID NO: 235)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACGGGGTACCCATGGTGGATCTACGCACGCTCGGCTACAGCCAGCAGCAACAGGAGAAGATCAAACCGAAGGTTCGTTCGACAGTGGCGCAGCACCACGAGGCACTGGTCGGCCATGGGTTTACACACGCGCACATCGTTGCGCTCAGCCAACACCCGGCAGCGTTAGGGACCGTCGCTGTCAAGTATCAGGACATGATCGCAGCGTTGCCAGAGGCGACACACGAAGCGATCGTTGGCGTCGGCAAACAGTGGTCCGGCGCACGCGCCCTGGAGGCCTTGCTCACGGTGGCGGGAGAGTTGAGAGGTCCACCGTTACAGTTGGACACAGGCCAACTTCTCAAGATTGCAAAACGTGGCGGCGTGACCGCAGTGGAGGCAGTGCATGCATGGCGCAATGCACTGACGGGGGCCCCCCTGAACCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGCACAGGTGGTGGCCATCGCCAGCAATATTGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGCGCCAGGCCCATGGCCTGACCCCGGAGCAGGTCGTGGCCATCGCCAGCAATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGCACAGGTGGTGGCCATCGCCAGCAATATTGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTCGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGACCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGACCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATGGCGGCGGCAGGCCGGCGCTGGAGAGCATTGTTGCCCAGTTATCTCGCCCTGATCCGTCGTTGGCCGCGTTAACCAACGACCACCTCGTCGCCTTGGCCTGCCTCGGCGGACGTCCTGCGCTGGATGCAGTGAAAAAGGGATTGCCGCACGCGCCGGCCTTGATCAAAAGAACCAATCGCCGTATTCCCGAACGCACATCCCATCGCGTTGCCGACCACGCGCAAGTGGTTCGCGTGCTGGGTTTTTTCCAGTGCCACTCCGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT >TALE13 +95 (SEQ ID NO: 236)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLRQAHGLTPEQVVAIASNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPSLAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS >TALE13 +95 (SEQ ID NO: 237)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACGGGGTACCCATGGTGGATCTACGCACGCTCGGCTACAGCCAGCAGCAACAGGAGAAGATCAAACCGAAGGTTCGTTCGACAGTGGCGCAGCACCACGAGGCACTGGTCGGCCATGGGTTTACACACGCGCACATCGTTGCGCTCAGCCAACACCCGGCAGCGTTAGGGACCGTCGCTGTCAAGTATCAGGACATGATCGCAGCGTTGCCAGAGGCGACACACGAAGCGATCGTTGGCGTCGGCAAACAGTGGTCCGGCGCACGCGCCCTGGAGGCCTTGCTCACGGTGGCGGGAGAGTTGAGAGGTCCACCGTTACAGTTGGACACAGGCCAACTTCTCAAGATTGCAAAACGTGGCGGCGTGACCGCAGTGGAGGCAGTGCATGCATGGCGCAATGCACTGACGGGGGCCCCCCTGAACCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGCACAGGTGGTGGCCATCGCCAGCAATATTGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGCGCCAGGCCCATGGCCTGACCCCGGAGCAGGTCGTGGCCATCGCCAGCAATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGCACAGGTGGTGGCCATCGCCAGCAATATTGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTCGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGACCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGACCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATGGCGGCGGCAGGCCGGCGCTGGAGAGCATTGTTGCCCAGTTATCTCGCCCTGATCCGTCGTTGGCCGCGTTAACCAACGACCACCTCGTCGCCTTGGCCTGCCTCGGCGGACGTCCTGCGCTGGATGCAGTGAAAAAGGGATTGCCGCACGCGCCGGCCTTGATCAAAAGAACCAATCGCCGTATTCCCGAACGCACATCCCATCGCGTTGCCGACCACGCGCAAGTGGTTCGCGTGCTGGGTTTTTTCCAGTGCCACTCCCACCCAGCGCAAGCATTTGATGACGCCATGACGCAGTTCGGGATGAGCGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT

2. TALEN Constructs and Protein Sequences Used for CCR5 Studies

Complete TALEN Construct Sequence, with Coding Sequence Underlined (SEQID NO:238):

GACTCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGAGCCAAGCTGACTAGCGTTTAAACTTAAGCTGATCCACTAGTCCAGTGTGGTGGAATTCGCCATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTATGGTGGACTTGAGGACACTCGGTTATTCGCAACAGCAACAGGAGAAAATCAAGCCTAAGGTCAGGAGCACCGTCGCGCAACACCACGAGGCGCTTGTGGGGCATGGCTTCACTCATGCGCATATTGTCGCGCTTTCACAGCACCCTGCGGCGCTTGGGACGGTGGCTGTCAAATACCAAGATATGATTGCGGCCCTGCCCGAAGCCACGCACGAGGCAATTGTAGGGGTCGGTAAACAGTGGTCGGGAGCGCGAGCACTTGAGGCGCTGCTGACTGTGGCGGGTGAGCTTAGGGGGCCTCCGCTCCAGCTCGACACCGGGCAGCTGCTGAAGATCGCGAAGAGAGGGGGAGTAACAGCGGTAGAGGCAGTGCACGCCTGGCGCAATGCGCTCACCGGGGCCCCCTTGAACCTGACCCCAGACCAGGTAGTCGCAATCGCCAACAATAACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCACATGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAATGGGGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCAACAACAACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGACTCACCCCAGACCAGGTAGTCGCAATCGCCAACAATAACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAACGGAGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCCATGATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAATATTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGGCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAATGGCGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCACATGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCCATGATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAACGGTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGTTTGACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACGCCTGCACAAGTGGTCGCCATCGCCTCCAATATTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGCCTGACACCCGAACAGGTGGTCGCCATTGCTAGCAACGGGGGAGGACGGCCAGCCTTGGAGTCCATCGTAGCCCAATTGTCCAGGCCCGATCCCGCGTTGGCTGCGTTAACGAATGACCATCTGGTGGCGTTGGCATGTCTTGGTGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCTTGATAACTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAATTGCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGGATGGCTTTCTCGCCGCCAAGGATCTGATGGCGCAGGGGATCAAGCTCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGAGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAATTATTAACGCTTACAATTTCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATAGCACGTGCTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGGCTTTTGCTGGCCTTTTGCTCACATGTTCTT

Complete Protein and Coding Sequence for Each CCR-5-Targeted TALEN:

To regenerate the sequence of each expression construct, replace theunderlined region of the above construct with each CDS shown below.

>CCR5 L161 (+28) (SEQ ID NO: 239)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS > CCR5 L161 (+28) (SEQ ID NO: 240)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTATGGTGGACTTGAGGACACTCGGTTATTCGCAACAGCAACAGGAGAAAATCAAGCCTAAGGTCAGGAGCACCGTCGCGCAACACCACGAGGCGCTTGTGGGGCATGGCTTCACTCATGCGCATATTGTCGCGCTTTCACAGCACCCTGCGGCGCTTGGGACGGTGGCTGTCAAATACCAAGATATGATTGCGGCCCTGCCCGAAGCCACGCACGAGGCAATTGTAGGGGTCGGTAAACAGTGGTCGGGAGCGCGAGCACTTGAGGCGCTGCTGACTGTGGCGGGTGAGCTTAGGGGGCCTCCGCTCCAGCTCGACACCGGGCAGCTGCTGAAGATCGCGAAGAGAGGGGGAGTAACAGCGGTAGAGGCAGTGCACGCCTGGCGCAATGCGCTCACCGGGGCCCCCTTGAACCTGACCCCAGACCAGGTAGTCGCAATCGCCAACAATAACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCACATGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAATGGGGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCAACAACAACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGACTCACCCCAGACCAGGTAGTCGCAATCGCCAACAATAACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAACGGAGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCCATGATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAATATTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGGCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAATGGCGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCACATGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCCATGATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAACGGTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGTTTGACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACGCCTGCACAAGTGGTCGCCATCGCCTCCAATATTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGCCTGACACCCGAACAGGTGGTCGCCATTGCTAGCAACGGGGGAGGACGGCCAGCCTTGGAGTCCATCGTAGCCCAATTGTCCAGGCCCGATCCCGCGTTGGCTGCGTTAACGAATGACCATCTGGTGGCGTTGGCATGTCTTGGTGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT > CCR5L161 (+63) (SEQ ID NO: 241)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS > CCR5L161 (+63) (SEQ ID NO: 242)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTATGGTGGACTTGAGGACACTCGGTTATTCGCAACAGCAACAGGAGAAAATCAAGCCTAAGGTCAGGAGCACCGTCGCGCAACACCACGAGGCGCTTGTGGGGCATGGCTTCACTCATGCGCATATTGTCGCGCTTTCACAGCACCCTGCGGCGCTTGGGACGGTGGCTGTCAAATACCAAGATATGATTGCGGCCCTGCCCGAAGCCACGCACGAGGCAATTGTAGGGGTCGGTAAACAGTGGTCGGGAGCGCGAGCACTTGAGGCGCTGCTGACTGTGGCGGGTGAGCTTAGGGGGCCTCCGCTCCAGCTCGACACCGGGCAGCTGCTGAAGATCGCGAAGAGAGGGGGAGTAACAGCGGTAGAGGCAGTGCACGCCTGGCGCAATGCGCTCACCGGGGCCCCCTTGAACCTGACCCCAGACCAGGTAGTCGCAATCGCCAACAATAACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCACATGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAATGGGGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCAACAACAACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGACTCACCCCAGACCAGGTAGTCGCAATCGCCAACAATAACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAACGGAGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCCATGATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAATATTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGGCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAATGGCGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCACATGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCCATGATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAACGGTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGTTTGACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACGCCTGCACAAGTGGTCGCCATCGCCTCCAATATTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGCCTGACACCCGAACAGGTGGTCGCCATTGCTAGCAACGGGGGAGGACGGCCAGCCTTGGAGTCCATCGTAGCCCAATTGTCCAGGCCCGATCCCGCGTTGGCTGCGTTAACGAATGACCATCTGGTGGCGTTGGCATGTCTTGGTGGACGACCCGCGCTCGATGCAGTCAAAAAGGGTCTGCCTCATGCTCCCGCATTGATCAAAAGAACCAACCGGCGGATTCCCGAGAGAACTTCCCATCGAGTCGCGGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT > CCR5L164 (+28) (SEQ ID NO: 243)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS > CCR5 L164 (+28) (SEQ ID NO: 244)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTATGGTGGACTTGAGGACACTCGGTTATTCGCAACAGCAACAGGAGAAAATCAAGCCTAAGGTCAGGAGCACCGTCGCGCAACACCACGAGGCGCTTGTGGGGCATGGCTTCACTCATGCGCATATTGTCGCGCTTTCACAGCACCCTGCGGCGCTTGGGACGGTGGCTGTCAAATACCAAGATATGATTGCGGCCCTGCCCGAAGCCACGCACGAGGCAATTGTAGGGGTCGGTAAACAGTGGTCGGGAGCGCGAGCACTTGAGGCGCTGCTGACTGTGGCGGGTGAGCTTAGGGGGCCTCCGCTCCAGCTCGACACCGGGCAGCTGCTGAAGATCGCGAAGAGAGGGGGAGTAACAGCGGTAGAGGCAGTGCACGCCTGGCGCAATGCGCTCACCGGGGCCCCCTTGAACCTGACCCCAGACCAGGTAGTCGCAATCGCCAACAATAACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGATCAAGTCGTGGCCATTGCAAATAATAACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAATGGGGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCCACGACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGACCATGGACTCACCCCAGACCAGGTAGTCGCAATCGCGTCGAACATTGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAACGGAGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCCATGATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCCACGACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGGCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAATGGCGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCACATGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAACATCGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAACGGTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGTTTGACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACGCCTGCACAAGTGGTCGCCATCGCCTCCCACGACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGCCTGACACCCGAACAGGTGGTCGCCATTGCTAGCAACGGGGGAGGACGGCCAGCCTTGGAGTCCATCGTAGCCCAATTGTCCAGGCCCGATCCCGCGTTGGCTGCGTTAACGAATGACCATCTGGTGGCGTTGGCATGTCTTGGTGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT > CCR5L164 (+63) (SEQ ID NO: 245)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS >CCR5L164 (+63) (SEQ ID NO: 246)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTATGGTGGACTTGAGGACACTCGGTTATTCGCAACAGCAACAGGAGAAAATCAAGCCTAAGGTCAGGAGCACCGTCGCGCAACACCACGAGGCGCTTGTGGGGCATGGCTTCACTCATGCGCATATTGTCGCGCTTTCACAGCACCCTGCGGCGCTTGGGACGGTGGCTGTCAAATACCAAGATATGATTGCGGCCCTGCCCGAAGCCACGCACGAGGCAATTGTAGGGGTCGGTAAACAGTGGTCGGGAGCGCGAGCACTTGAGGCGCTGCTGACTGTGGCGGGTGAGCTTAGGGGGCCTCCGCTCCAGCTCGACACCGGGCAGCTGCTGAAGATCGCGAAGAGAGGGGGAGTAACAGCGGTAGAGGCAGTGCACGCCTGGCGCAATGCGCTCACCGGGGCCCCCTTGAACCTGACCCCAGACCAGGTAGTCGCAATCGCCAACAATAACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGATCAAGTCGTGGCCATTGCAAATAATAACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAATGGGGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCCACGACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGACTCACCCCAGACCAGGTAGTCGCAATCGCGTCGAACATTGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAACGGAGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCCATGATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCCACGACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGGCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAATGGCGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCACATGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAACATCGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAACGGTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGTTTGACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACGCCTGCACAAGTGGTCGCCATCGCCTCCCACGACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGCCTGACACCCGAACAGGTGGTCGCCATTGCTAGCAACGGGGGAGGACGGCCAGCCTTGGAGTCCATCGTAGCCCAATTGTCCAGGCCCGATCCCGCGTTGGCTGCGTTAACGAATGACCATCTGGTGGCGTTGGCATGTCTTGGTGGACGACCCGCGCTCGATGCAGTCAAAAAGGGTCTGCCTCATGCTCCCGCATTGATCAAAAGAACCAACCGGCGGATTCCCGAGAGAACTTCCCATCGAGTCGCGGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT > CCR5L167 (+28) (SEQ ID NO: 247)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS > CCR5 L167 (+28) (SEQ ID NO: 248)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTATGGTGGACTTGAGGACACTCGGTTATTCGCAACAGCAACAGGAGAAAATCAAGCCTAAGGTCAGGAGCACCGTCGCGCAACACCACGAGGCGCTTGTGGGGCATGGCTTCACTCATGCGCATATTGTCGCGCTTTCACAGCACCCTGCGGCGCTTGGGACGGTGGCTGTCAAATACCAAGATATGATTGCGGCCCTGCCCGAAGCCACGCACGAGGCAATTGTAGGGGTCGGTAAACAGTGGTCGGGAGCGCGAGCACTTGAGGCGCTGCTGACTGTGGCGGGTGAGCTTAGGGGGCCTCCGCTCCAGCTCGACACCGGGCAGCTGCTGAAGATCGCGAAGAGAGGGGGAGTAACAGCGGTAGAGGCAGTGCACGCCTGGCGCAATGCGCTCACCGGGGCCCCCTTGAACCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAATATCGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAATGGGGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCCACGACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGACTCACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAACGGAGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCCATGATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAATATTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGGCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAATGGCGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCACATGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCCATGATGGAGGGAAACAGGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAACGGTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGTTTGACCCCAGACCAGGTAGTCGCAATCGCCAACAATAACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACGCCTGCACAAGTGGTCGCCATCGCCTCCAATATTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGCCTGACACCCGAACAGGTGGTCGCCATTGCTAGCAACGGGGGAGGACGGCCAGCCTTGGAGTCCATCGTAGCCCAATTGTCCAGGCCCGATCCCGCGTTGGCTGCGTTAACGAATGACCATCTGGTGGCGTTGGCATGTCTTGGTGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT > CCR5L167 (+63) (SEQ ID NO: 249)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS > CCR5L167 (+63) (SEQ ID NO: 250)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTATGGTGGACTTGAGGACACTCGGTTATTCGCAACAGCAACAGGAGAAAATCAAGCCTAAGGTCAGGAGCACCGTCGCGCAACACCACGAGGCGCTTGTGGGGCATGGCTTCACTCATGCGCATATTGTCGCGCTTTCACAGCACCCTGCGGCGCTTGGGACGGTGGCTGTCAAATACCAAGATATGATTGCGGCCCTGCCCGAAGCCACGCACGAGGCAATTGTAGGGGTCGGTAAACAGTGGTCGGGAGCGCGAGCACTTGAGGCGCTGCTGACTGTGGCGGGTGAGCTTAGGGGGCCTCCGCTCCAGCTCGACACCGGGCAGCTGCTGAAGATCGCGAAGAGAGGGGGAGTAACAGCGGTAGAGGCAGTGCACGCCTGGCGCAATGCGCTCACCGGGGCCCCCTTGAACCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAATATCGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAATGGGGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCCACGACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGACTCACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAACGGAGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCCATGATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAATATTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGGCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAATGGCGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCACATGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCCATGATGGAGGGAAACAGGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAACGGTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGTTTGACCCCAGACCAGGTAGTCGCAATCGCCAACAATAACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACGCCTGCACAAGTGGTCGCCATCGCCTCCAATATTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGCCTGACACCCGAACAGGTGGTCGCCATTGCTAGCAACGGGGGAGGACGGCCAGCCTTGGAGTCCATCGTAGCCCAATTGTCCAGGCCCGATCCCGCGTTGGCTGCGTTAACGAATGACCATCTGGTGGCGTTGGCATGTCTTGGTGGACGACCCGCGCTCGATGCAGTCAAAAAGGGTCTGCCTCATGCTCCCGCATTGATCAAAAGAACCAACCGGCGGATTCCCGAGAGAACTTCCCATCGAGTCGCGGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT > CCR5L172 (+28) (SEQ ID NO: 251)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVARKFNNGEINFRS > CCR5L172 (+28) (SEQ ID NO: 252)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTATGGTGGACTTGAGGACACTCGGTTATTCGCAACAGCAACAGGAGAAAATCAAGCCTAAGGTCAGGAGCACCGTCGCGCAACACCACGAGGCGCTTGTGGGGCATGGCTTCACTCATGCGCATATTGTCGCGCTTTCACAGCACCCTGCGGCGCTTGGGACGGTGGCTGTCAAATACCAAGATATGATTGCGGCCCTGCCCGAAGCCACGCACGAGGCAATTGTAGGGGTCGGTAAACAGTGGTCGGGAGCGCGAGCACTTGAGGCGCTGCTGACTGTGGCGGGTGAGCTTAGGGGGCCTCCGCTCCAGCTCGACACCGGGCAGCTGCTGAAGATCGCGAAGAGAGGGGGAGTAACAGCGGTAGAGGCAGTGCACGCCTGGCGCAATGCGCTCACCGGGGCCCCCTTGAACCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCACATGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAATGGGGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCCACGACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGACTCACCCCAGACCAGGTAGTCGCAATCGCGTCGAACATTGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAACGGAGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCCATGATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCCACGACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGGCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAATGGCGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGATCAAGTCGTGGCCATTGCAAATAATAACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAACATCGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAACGGTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGTTTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAACATTGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCTAATATCGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAACATCGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCCACGACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGCCTGACACCCGAACAGGTGGTCGCCATTGCTAGCAACGGGGGAGGACGGCCAGCCTTGGAGTCCATCGTAGCCCAATTGTCCAGGCCCGATCCCGCGTTGGCTGCGTTAACGAATGACCATCTGGTGGCGTTGGCATGTCTTGGTGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT > CCR5L172 (+63) (SEQ ID NO: 253)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS > CCR5 L172 (+63) (SEQ ID NO: 254)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTATGGTGGACTTGAGGACACTCGGTTATTCGCAACAGCAACAGGAGAAAATCAAGCCTAAGGTCAGGAGCACCGTCGCGCAACACCACGAGGCGCTTGTGGGGCATGGCTTCACTCATGCGCATATTGTCGCGCTTTCACAGCACCCTGCGGCGCTTGGGACGGTGGCTGTCAAATACCAAGATATGATTGCGGCCCTGCCCGAAGCCACGCACGAGGCAATTGTAGGGGTCGGTAAACAGTGGTCGGGAGCGCGAGCACTTGAGGCGCTGCTGACTGTGGCGGGTGAGCTTAGGGGGCCTCCGCTCCAGCTCGACACCGGGCAGCTGCTGAAGATCGCGAAGAGAGGGGGAGTAACAGCGGTAGAGGCAGTGCACGCCTGGCGCAATGCGCTCACCGGGGCCCCCTTGAACCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCACATGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAATGGGGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCCACGACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGACTCACCCCAGACCAGGTAGTCGCAATCGCGTCGAACATTGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAACGGAGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCCATGATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCCACGACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGGCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAATGGCGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGATCAAGTCGTGGCCATTGCAAATAATAACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAACATCGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAACGGTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGTTTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAACATTGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCTAATATCGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAACATCGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCCACGACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGCCTGACACCCGAACAGGTGGTCGCCATTGCTAGCAACGGGGGAGGACGGCCAGCCTTGGAGTCCATCGTAGCCCAATTGTCCAGGCCCGATCCCGCGTTGGCTGCGTTAACGAATGACCATCTGGTGGCGTTGGCATGTCTTGGTGGACGACCCGCGCTCGATGCAGTCAAAAAGGGTCTGCCTCATGCTCCCGCATTGATCAAAAGAACCAACCGGCGGATTCCCGAGAGAACTTCCCATCGAGTCGCGGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT >CCR5 R175 (+28) (SEQ ID NO: 255)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS >CCR5 R175 (+28) (SEQ ID NO: 256)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTATGGTGGACTTGAGGACACTCGGTTATTCGCAACAGCAACAGGAGAAAATCAAGCCTAAGGTCAGGAGCACCGTCGCGCAACACCACGAGGCGCTTGTGGGGCATGGCTTCACTCATGCGCATATTGTCGCGCTTTCACAGCACCCTGCGGCGCTTGGGACGGTGGCTGTCAAATACCAAGATATGATTGCGGCCCTGCCCGAAGCCACGCACGAGGCAATTGTAGGGGTCGGTAAACAGTGGTCGGGAGCGCGAGCACTTGAGGCGCTGCTGACTGTGGCGGGTGAGCTTAGGGGGCCTCCGCTCCAGCTCGACACCGGGCAGCTGCTGAAGATCGCGAAGAGAGGGGGAGTAACAGCGGTAGAGGCAGTGCACGCCTGGCGCAATGCGCTCACCGGGGCCCCCTTGAACCTGACCCCAGACCAGGTAGTCGCAATCGCCAACAATAACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCACATGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAATGGGGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCCACGACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGACTCACCCCAGACCAGGTAGTCGCAATCGCGTCGAATGGCGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAACGGAGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCCATGATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAATATTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGGCTGACCCCAGACCAGGTAGTCGCAATCGCCAACAATAACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCACATGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCCATGATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAACGGTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGTTTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAATGGCGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACGCCTGCACAAGTGGTCGCCATCGCCTCCAACGGTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGCCTGACACCCGAACAGGTGGTCGCCATTGCTAGCAACGGGGGAGGACGGCCAGCCTTGGAGTCCATCGTAGCCCAATTGTCCAGGCCCGATCCCGCGTTGGCTGCGTTAACGAATGACCATCTGGTGGCGTTGGCATGTCTTGGTGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT > CCR5R175 (+63) (SEQ ID NO: 257)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS > CCR5R175 (+63) (SEQ ID NO: 258)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTATGGTGGACTTGAGGACACTCGGTTATTCGCAACAGCAACAGGAGAAAATCAAGCCTAAGGTCAGGAGCACCGTCGCGCAACACCACGAGGCGCTTGTGGGGCATGGCTTCACTCATGCGCATATTGTCGCGCTTTCACAGCACCCTGCGGCGCTTGGGACGGTGGCTGTCAAATACCAAGATATGATTGCGGCCCTGCCCGAAGCCACGCACGAGGCAATTGTAGGGGTCGGTAAACAGTGGTCGGGAGCGCGAGCACTTGAGGCGCTGCTGACTGTGGCGGGTGAGCTTAGGGGGCCTCCGCTCCAGCTCGACACCGGGCAGCTGCTGAAGATCGCGAAGAGAGGGGGAGTAACAGCGGTAGAGGCAGTGCACGCCTGGCGCAATGCGCTCACCGGGGCCCCCTTGAACCTGACCCCAGACCAGGTAGTCGCAATCGCCAACAATAACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCACATGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAATGGGGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCCACGACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGACTCACCCCAGACCAGGTAGTCGCAATCGCGTCGAATGGCGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAACGGAGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCCATGATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAATATTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGGCTGACCCCAGACCAGGTAGTCGCAATCGCCAACAATAACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCACATGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCCATGATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAACGGTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGTTTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAATGGCGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACGCCTGCACAAGTGGTCGCCATCGCCTCCAACGGTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGCTTGACACCCGAACAGGTGGTCGCCATTGCTAGCAACGGGGGAGGACGGCCAGCCTTGGAGTCCATCGTAGCCCAATTGTCCAGGCCCGATCCCGCGTTGGCTGCGTTAACGAATGACCATCTGGTGGCGTTGGCATGTCTTGGTGGACGACCCGCGCTCGATGCAGTCAAAAAGGGTCTGCCTCATGCTCCCGCATTGATCAAAAGAACCAACCGGCGGATTCCCGAGAGAACTTCCCATCGAGTCGCGGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT > CCR5R177 (+28) (SEQ ID NO: 259)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS > CCR5 R177(+28) (SEQ ID NO: 260)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTATGGTGGACTTGAGGACACTCGGTTATTCGCAACAGCAACAGGAGAAAATCAAGCCTAAGGTCAGGAGCACCGTCGCGCAACACCACGAGGCGCTTGTGGGGCATGGCTTCACTCATGCGCATATTGTCGCGCTTTCACAGCACCCTGCGGCGCTTGGGACGGTGGCTGTCAAATACCAAGATATGATTGCGGCCCTGCCCGAAGCCACGCACGAGGCAATTGTAGGGGTCGGTAAACAGTGGTCGGGAGCGCGAGCACTTGAGGCGCTGCTGACTGTGGCGGGTGAGCTTAGGGGGCCTCCGCTCCAGCTCGACACCGGGCAGCTGCTGAAGATCGCGAAGAGAGGGGGAGTAACAGCGGTAGAGGCAGTGCACGCCTGGCGCAATGCGCTCACCGGGGCCCCCTTGAACCTGACCCCAGACCAGGTAGTCGCAATCGCCAACAATAACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCACATGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAATGGGGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCCACGACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGACTCACCCCAGACCAGGTAGTCGCAATCGCGTCGAATGGCGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAACGGAGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCCATGATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAATATTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGGCTGACCCCAGACCAGGTAGTCGCAATCGCCAACAATAACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCACATGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCCATGATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAACGGTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGCCTGACACCCGAACAGGTGGTCGCCATTGCTAGCAACGGGGGAGGACGGCCAGCCTTGGAGTCCATCGTAGCCCAATTGTCCAGGCCCGATCCCGCGTTGGCTGCGTTAACGAATGACCATCTGGTGGCGTTGGCATGTCTTGGTGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT > CCR5R177 (+63) (SEQ ID NO: 261)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS > CCR5R177 (+63) (SEQ ID NO: 262)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTATGGTGGACTTGAGGACACTCGGTTATTCGCAACAGCAACAGGAGAAAATCAAGCCTAAGGTCAGGAGCACCGTCGCGCAACACCACGAGGCGCTTGTGGGGCATGGCTTCACTCATGCGCATATTGTCGCGCTTTCACAGCACCCTGCGGCGCTTGGGACGGTGGCTGTCAAATACCAAGATATGATTGCGGCCCTGCCCGAAGCCACGCACGAGGCAATTGTAGGGGTCGGTAAACAGTGGTCGGGAGCGCGAGCACTTGAGGCGCTGCTGACTGTGGCGGGTGAGCTTAGGGGGCCTCCGCTCCAGCTCGACACCGGGCAGCTGCTGAAGATCGCGAAGAGAGGGGGAGTAACAGCGGTAGAGGCAGTGCACGCCTGGCGCAATGCGCTCACCGGGGCCCCCTTGAACCTGACCCCAGACCAGGTAGTCGCAATCGCCAACAATAACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCACATGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAATGGGGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCCACGACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGACTCACCCCAGACCAGGTAGTCGCAATCGCGTCGAATGGCGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAACGGAGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCCATGATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAATATTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGGCTGACCCCAGACCAGGTAGTCGCAATCGCCAACAATAACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCACATGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCCATGATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAACGGTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGCCTGACACCCGAACAGGTGGTCGCCATTGCTAGCAACGGGGGAGGACGGCCAGCCTTGGAGTCCATCGTAGCCCAATTGTCCAGGCCCGATCCCGCGTTGGCTGCGTTAACGAATGACCATCTGGTGGCGTTGGCATGTCTTGGTGGACGACCCGCGCTCGATGCAGTCAAAAAGGGTCTGCCTCATGCTCCCGCATTGATCAAAAGAACCAACCGGCGGATTCCCGAGAGAACTTCCCATCGAGTCGCGGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT > CCR5R178 (+28) (SEQ ID NO: 263)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS > CCR5 R178 (+28) (SEQ ID NO: 264)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTATGGTGGACTTGAGGACACTCGGTTATTCGCAACAGCAACAGGAGAAAATCAAGCCTAAGGTCAGGAGCACCGTCGCGCAACACCACGAGGCGCTTGTGGGGCATGGCTTCACTCATGCGCATATTGTCGCGCTTTCACAGCACCCTGCGGCGCTTGGGACGGTGGCTGTCAAATACCAAGATATGATTGCGGCCCTGCCCGAAGCCACGCACGAGGCAATTGTAGGGGTCGGTAAACAGTGGTCGGGAGCGCGAGCACTTGAGGCGCTGCTGACTGTGGCGGGTGAGCTTAGGGGGCCTCCGCTCCAGCTCGACACCGGGCAGCTGCTGAAGATCGCGAAGAGAGGGGGAGTAACAGCGGTAGAGGCAGTGCACGCCTGGCGCAATGCGCTCACCGGGGCCCCCTTGAACCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAATATCGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAATGGGGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCAACAACAACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGACTCACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAACGGAGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCCATGATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAACGGTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGGCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAATGGCGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCACATGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAACATCGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCAACAACAACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGTTTGACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACGCCTGCACAAGTGGTCGCCATCGCCTCCCACGACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGCCTGACACCCGAACAGGTGGTCGCCATTGCTAGCAACGGGGGAGGACGGCCAGCCTTGGAGTCCATCGTAGCCCAATTGTCCAGGCCCGATCCCGCGTTGGCTGCGTTAACGAATGACCATCTGGTGGCGTTGGCATGTCTTGGTGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT > CCR5R178 (+63) (SEQ ID NO: 265)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS > CCR5R178 (+63) (SEQ ID NO: 266)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTATGGTGGACTTGAGGACACTCGGTTATTCGCAACAGCAACAGGAGAAAATCAAGCCTAAGGTCAGGAGCACCGTCGCGCAACACCACGAGGCGCTTGTGGGGCATGGCTTCACTCATGCGCATATTGTCGCGCTTTCACAGCACCCTGCGGCGCTTGGGACGGTGGCTGTCAAATACCAAGATATGATTGCGGCCCTGCCCGAAGCCACGCACGAGGCAATTGTAGGGGTCGGTAAACAGTGGTCGGGAGCGCGAGCACTTGAGGCGCTGCTGACTGTGGCGGGTGAGCTTAGGGGGCCTCCGCTCCAGCTCGACACCGGGCAGCTGCTGAAGATCGCGAAGAGAGGGGGAGTAACAGCGGTAGAGGCAGTGCACGCCTGGCGCAATGCGCTCACCGGGGCCCCCTTGAACCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAATATCGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAATGGGGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCAACAACAACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGACTCACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAACGGAGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCCATGATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAACGGTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGGCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAATGGCGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCACATGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAACATCGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCAACAACAACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGTTTGACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACGCCTGCACAAGTGGTCGCCATCGCCTCCCACGACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGCCTGACACCCGAACAGGTGGTCGCCATTGCTAGCAACGGGGGAGGACGGCCAGCCTTGGAGTCCATCGTAGCCCAATTGTCCAGGCCCGATCCCGCGTTGGCTGCGTTAACGAATGACCATCTGGTGGCGTTGGCATGTCTTGGTGGACGACCCGCGCTCGATGCAGTCAAAAAGGGTCTGCCTCATGCTCCCGCATTGATCAAAAGAACCAACCGGCGGATTCCCGAGAGAACTTCCCATCGAGTCGCGGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT > CCR5R185 (+28) (SEQ ID NO: 267)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVARKFNNGEINFRS > CCR5R185 (+28) (SEQ ID NO: 268)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTATGGTGGACTTGAGGACACTCGGTTATTCGCAACAGCAACAGGAGAAAATCAAGCCTAAGGTCAGGAGCACCGTCGCGCAACACCACGAGGCGCTTGTGGGGCATGGCTTCACTCATGCGCATATTGTCGCGCTTTCACAGCACCCTGCGGCGCTTGGGACGGTGGCTGTCAAATACCAAGATATGATTGCGGCCCTGCCCGAAGCCACGCACGAGGCAATTGTAGGGGTCGGTAAACAGTGGTCGGGAGCGCGAGCACTTGAGGCGCTGCTGACTGTGGCGGGTGAGCTTAGGGGGCCTCCGCTCCAGCTCGACACCGGGCAGCTGCTGAAGATCGCGAAGAGAGGGGGAGTAACAGCGGTAGAGGCAGTGCACGCCTGGCGCAATGCGCTCACCGGGGCCCCCTTGAACCTGACCCCAGACCAGGTAGTCGCAATCGCCAACAATAACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAACGGAGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCCATGATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAATATTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGACTCACCCCAGACCAGGTAGTCGCAATCGCCAACAATAACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAACGGAGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCCATGATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAATATTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGGCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAATGGCGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGATCAAGTCGTGGCCATTGCAAATAATAACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCCATGATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAACGGTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGTTTGACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTACTGTGCCAGGATCATGGCCTGACACCCGAACAGGTGGTCGCCATTGCTAGCAACGGGGGAGGACGGCCAGCCTTGGAGTCCATCGTAGCCCAATTGTCCAGGCCCGATCCCGCGTTGGCTGCGTTAACGAATGACCATCTGGTGGCGTTGGCATGTCTTGGTGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT > CCR5R185 (+63) (SEQ ID NO: 269)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS > CCR5 R185 (+63) (SEQ ID NO: 270)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTATGGTGGACTTGAGGACACTCGGTTATTCGCAACAGCAACAGGAGAAAATCAAGCCTAAGGTCAGGAGCACCGTCGCGCAACACCACGAGGCGCTTGTGGGGCATGGCTTCACTCATGCGCATATTGTCGCGCTTTCACAGCACCCTGCGGCGCTTGGGACGGTGGCTGTCAAATACCAAGATATGATTGCGGCCCTGCCCGAAGCCACGCACGAGGCAATTGTAGGGGTCGGTAAACAGTGGTCGGGAGCGCGAGCACTTGAGGCGCTGCTGACTGTGGCGGGTGAGCTTAGGGGGCCTCCGCTCCAGCTCGACACCGGGCAGCTGCTGAAGATCGCGAAGAGAGGGGGAGTAACAGCGGTAGAGGCAGTGCACGCCTGGCGCAATGCGCTCACCGGGGCCCCCTTGAACCTGACCCCAGACCAGGTAGTCGCAATCGCCAACAATAACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAACGGAGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCCATGATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAATATTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGACTCACCCCAGACCAGGTAGTCGCAATCGCCAACAATAACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAACGGAGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCCATGATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAATATTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGGCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAATGGCGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGATCAAGTCGTGGCCATTGCAAATAATAACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCCATGATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAACGGTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGTTTGACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTACTGTGCCAGGATCATGGCCTGACACCCGAACAGGTGGTCGCCATTGCTAGCAACGGGGGAGGACGGCCAGCCTTGGAGTCCATCGTAGCCCAATTGTCCAGGCCCGATCCCGCGTTGGCTGCGTTAACGAATGACCATCTGGTGGCGTTGGCATGTCTTGGTGGACGACCCGCGCTCGATGCAGTCAAAAAGGGTCTGCCTCATGCTCCCGCATTGATCAAAAGAACCAACCGGCGGATTCCCGAGAGAACTTCCCATCGAGTCGCGGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT > CCR5L532 (+28) (SEQ ID NO: 271)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS > CCR5 L532(+28) (SEQ ID NO: 272)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTATGGTGGACTTGAGGACACTCGGTTATTCGCAACAGCAACAGGAGAAAATCAAGCCTAAGGTCAGGAGCACCGTCGCGCAACACCACGAGGCGCTTGTGGGGCATGGCTTCACTCATGCGCATATTGTCGCGCTTTCACAGCACCCTGCGGCGCTTGGGACGGTGGCTGTCAAATACCAAGATATGATTGCGGCCCTGCCCGAAGCCACGCACGAGGCAATTGTAGGGGTCGGTAAACAGTGGTCGGGAGCGCGAGCACTTGAGGCGCTGCTGACTGTGGCGGGTGAGCTTAGGGGGCCTCCGCTCCAGCTCGACACCGGGCAGCTGCTGAAGATCGCGAAGAGAGGGGGAGTAACAGCGGTAGAGGCAGTGCACGCCTGGCGCAATGCGCTCACCGGGGCCCCCTTGAACCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAACGGAGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAATGGGGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCCACGACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGACTCACCCCAGACCAGGTAGTCGCAATCGCGTCGAACATTGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAACGGAGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAATGGGGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAATATTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGGCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAATATCGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCCATGATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCCACGACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGCCTGACACCCGAACAGGTGGTCGCCATTGCTAGCAACGGGGGAGGACGGCCAGCCTTGGAGTCCATCGTAGCCCAATTGTCCAGGCCCGATCCCGCGTTGGCTGCGTTAACGAATGACCATCTGGTGGCGTTGGCATGTCTTGGTGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT > CCR5L532 (+63) (SEQ ID NO: 273)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVASTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS > CCR5L532 (+63) (SEQ ID NO: 274)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTATGGTGGACTTGAGGACACTCGGTTATTCGCAACAGCAACAGGAGAAAATCAAGCCTAAGGTCAGGAGCACCGTCGCGCAACACCACGAGGCGCTTGTGGGGCATGGCTTCACTCATGCGCATATTGTCGCGCTTTCACAGCACCCTGCGGCGCTTGGGACGGTGGCTGTCAAATACCAAGATATGATTGCGGCCCTGCCCGAAGCCACGCACGAGGCAATTGTAGGGGTCGGTAAACAGTGGTCGGGAGCGCGAGCACTTGAGGCGCTGCTGACTGTGGCGGGTGAGCTTAGGGGGCCTCCGCTCCAGCTCGACACCGGGCAGCTGCTGAAGATCGCGAAGAGAGGGGGAGTAACAGCGGTAGAGGCAGTGCACGCCTGGCGCAATGCGCTCACCGGGGCCCCCTTGAACCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAACGGAGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAATGGGGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCCACGACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGACTCACCCCAGACCAGGTAGTCGCAATCGCGTCGAACATTGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAACGGAGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAATGGGGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAATATTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGGCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAATATCGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCCATGATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCCACGACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGCCTGACACCCGAACAGGTGGTCGCCATTGCTAGCAACGGGGGAGGACGGCCAGCCTTGGAGTCCATCGTAGCCCAATTGTCCAGGCCCGATCCCGCGTTGGCTGCGTTAACGAATGACCATCTGGTGGCGTTGGCATGTCTTGGTGGACGACCCGCGCTCGATGCAGTCAAAAAGGGTCTGCCTCATGCTCCCGCATTGATCAAAAGAACCAACCGGCGGATTCCCGAGAGAACTTCCCATCGAGTCGCGGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT > CCR5L538 (+28) (SEQ ID NO: 275)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS > CCR5L538 (+28) (SEQ ID NO: 276)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTATGGTGGACTTGAGGACACTCGGTTATTCGCAACAGCAACAGGAGAAAATCAAGCCTAAGGTCAGGAGCACCGTCGCGCAACACCACGAGGCGCTTGTGGGGCATGGCTTCACTCATGCGCATATTGTCGCGCTTTCACAGCACCCTGCGGCGCTTGGGACGGTGGCTGTCAAATACCAAGATATGATTGCGGCCCTGCCCGAAGCCACGCACGAGGCAATTGTAGGGGTCGGTAAACAGTGGTCGGGAGCGCGAGCACTTGAGGCGCTGCTGACTGTGGCGGGTGAGCTTAGGGGGCCTCCGCTCCAGCTCGACACCGGGCAGCTGCTGAAGATCGCGAAGAGAGGGGGAGTAACAGCGGTAGAGGCAGTGCACGCCTGGCGCAATGCGCTCACCGGGGCCCCCTTGAACCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAATGGCGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCACATGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAACATCGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAACGGTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGACTCACCCCAGACCAGGTAGTCGCAATCGCGTCGAATGGCGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAATATCGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCCATGATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAATATTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGACCATGGGCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCACATGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAATGGGGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCAACAACAACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGTTTGACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAATATCGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAATAACAATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCCACGACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGCCTGACACCCGAACAGGTGGTCGCCATTGCTAGCAACGGGGGAGGACGGCCAGCCTTGGAGTCCATCGTAGCCCAATTGTCCAGGCCCGATCCCGCGTTGGCTGCGTTAACGAATGACCATCTGGTGGCGTTGGCATGTCTTGGTGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT > CCR5L538 (+63) (SEQ ID NO: 277)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS > CCR5 L538 (+63) (SEQ ID NO: 278)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTATGGTGGACTTGAGGACACTCGGTTATTCGCAACAGCAACAGGAGAAAATCAAGCCTAAGGTCAGGAGCACCGTCGCGCAACACCACGAGGCGCTTGTGGGGCATGGCTTCACTCATGCGCATATTGTCGCGCTTTCACAGCACCCTGCGGCGCTTGGGACGGTGGCTGTCAAATACCAAGATATGATTGCGGCCCTGCCCGAAGCCACGCACGAGGCAATTGTAGGGGTCGGTAAACAGTGGTCGGGAGCGCGAGCACTTGAGGCGCTGCTGACTGTGGCGGGTGAGCTTAGGGGGCCTCCGCTCCAGCTCGACACCGGGCAGCTGCTGAAGATCGCGAAGAGAGGGGGAGTAACAGCGGTAGAGGCAGTGCACGCCTGGCGCAATGCGCTCACCGGGGCCCCCTTGAACCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAATGGCGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCACATGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAACATCGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAACGGTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGACTCACCCCAGACCAGGTAGTCGCAATCGCGTCGAATGGCGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAATATCGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCCATGATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAATATTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGGCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCACATGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAATGGGGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCAACAACAACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGTTTGACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAATATCGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAATAACAATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCCACGACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGCCTGACACCCGAACAGGTGGTCGCCATTGCTAGCAACGGGGGAGGACGGCCAGCCTTGGAGTCCATCGTAGCCCAATTGTCCAGGCCCGATCCCGCGTTGGCTGCGTTAACGAATGACCATCTGGTGGCGTTGGCATGTCTTGGTGGACGACCCGCGCTCGATGCAGTCAAAAAGGGTCTGCCTCATGCTCCCGCATTGATCAAAAGAACCAACCGGCGGATTCCCGAGAGAACTTCCCATCGAGTCGCGGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT > CCR5 L540 (+28) (SEQ ID NO: 279)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS > CCR5L540 (+28) (SEQ ID NO: 280)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTATGGTGGACTTGAGGACACTCGGTTATTCGCAACAGCAACAGGAGAAAATCAAGCCTAAGGTCAGGAGCACCGTCGCGCAACACCACGAGGCGCTTGTGGGGCATGGCTTCACTCATGCGCATATTGTCGCGCTTTCACAGCACCCTGCGGCGCTTGGGACGGTGGCTGTCAAATACCAAGATATGATTGCGGCCCTGCCCGAAGCCACGCACGAGGCAATTGTAGGGGTCGGTAAACAGTGGTCGGGAGCGCGAGCACTTGAGGCGCTGCTGACTGTGGCGGGTGAGCTTAGGGGGCCTCCGCTCCAGCTCGACACCGGGCAGCTGCTGAAGATCGCGAAGAGAGGGGGAGTAACAGCGGTAGAGGCAGTGCACGCCTGGCGCAATGCGCTCACCGGGGCCCCCTTGAACCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAACATTGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCACATGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAACATCGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCCACGACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGACTCACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAACGGAGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAATAACAATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCCACGACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGGCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAACATTGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGATCAAGTCGTGGCCATTGCAAATAATAACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCCATGATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAACGGTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGTTTGACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTACTGTGCCAGGATCATGGCCTGACACCCGAACAGGTGGTCGCCATTGCTAGCAACGGGGGAGGACGGCCAGCCTTGGAGTCCATCGTAGCCCAATTGTCCAGGCCCGATCCCGCGTTGGCTGCGTTAACGAATGACCATCTGGTGGCGTTGGCATGTCTTGGTGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT > CCR5L540 (+63) (SEQ ID NO: 281)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS > CCR5 L540 (+63) (SEQ ID NO: 282)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTATGGTGGACTTGAGGACACTCGGTTATTCGCAACAGCAACAGGAGAAAATCAAGCCTAAGGTCAGGAGCACCGTCGCGCAACACCACGAGGCGCTTGTGGGGCATGGCTTCACTCATGCGCATATTGTCGCGCTTTCACAGCACCCTGCGGCGCTTGGGACGGTGGCTGTCAAATACCAAGATATGATTGCGGCCCTGCCCGAAGCCACGCACGAGGCAATTGTAGGGGTCGGTAAACAGTGGTCGGGAGCGCGAGCACTTGAGGCGCTGCTGACTGTGGCGGGTGAGCTTAGGGGGCCTCCGCTCCAGCTCGACACCGGGCAGCTGCTGAAGATCGCGAAGAGAGGGGGAGTAACAGCGGTAGAGGCAGTGCACGCCTGGCGCAATGCGCTCACCGGGGCCCCCTTGAACCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAACATTGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCACATGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAACATCGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCCACGACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGACTCACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAACGGAGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAATAACAATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCCACGACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGGCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAACATTGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGATCAAGTCGTGGCCATTGCAAATAATAACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCCATGATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAACGGTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGTTTGACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTACTGTGCCAGGATCATGGCCTGACACCCGAACAGGTGGTCGCCATTGCTAGCAACGGGGGAGGACGGCCAGCCTTGGAGTCCATCGTAGCCCAATTGTCCAGGCCCGATCCCGCGTTGGCTGCGTTAACGAATGACCATCTGGTGGCGTTGGCATGTCTTGGTGGACGACCCGCGCTCGATGCAGTCAAAAAGGGTCTGCCTCATGCTCCCGCATTGATCAAAAGAACCAACCGGCGGATTCCCGAGAGAACTTCCCATCGAGTCGCGGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT > CCR5L543 (+28) (SEQ ID NO: 283)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS > CCR5L543 (+28) (SEQ ID NO: 284)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTATGGTGGACTTGAGGACACTCGGTTATTCGCAACAGCAACAGGAGAAAATCAAGCCTAAGGTCAGGAGCACCGTCGCGCAACACCACGAGGCGCTTGTGGGGCATGGCTTCACTCATGCGCATATTGTCGCGCTTTCACAGCACCCTGCGGCGCTTGGGACGGTGGCTGTCAAATACCAAGATATGATTGCGGCCCTGCCCGAAGCCACGCACGAGGCAATTGTAGGGGTCGGTAAACAGTGGTCGGGAGCGCGAGCACTTGAGGCGCTGCTGACTGTGGCGGGTGAGCTTAGGGGGCCTCCGCTCCAGCTCGACACCGGGCAGCTGCTGAAGATCGCGAAGAGAGGGGGAGTAACAGCGGTAGAGGCAGTGCACGCCTGGCGCAATGCGCTCACCGGGGCCCCCTTGAACCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAACATTGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCACATGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAACATCGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCCACGACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGACTCACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAACGGAGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAATAACAATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCCACGACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGGCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAACATTGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGATCAAGTCGTGGCCATTGCAAATAATAACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCCATGATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAACGGTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGTTTGACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAACGGAGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCCATGATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAATATTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGCCTGACACCCGAACAGGTGGTCGCCATTGCTAGCAACGGGGGAGGACGGCCAGCCTTGGAGTCCATCGTAGCCCAATTGTCCAGGCCCGATCCCGCGTTGGCTGCGTTAACGAATGACCATCTGGTGGCGTTGGCATGTCTTGGTGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT > CCR5L543 (+63) (SEQ ID NO: 285)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS > CCR5 L543 (+63) (SEQ ID NO: 286)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTATGGTGGACTTGAGGACACTCGGTTATTCGCAACAGCAACAGGAGAAAATCAAGCCTAAGGTCAGGAGCACCGTCGCGCAACACCACGAGGCGCTTGTGGGGCATGGCTTCACTCATGCGCATATTGTCGCGCTTTCACAGCACCCTGCGGCGCTTGGGACGGTGGCTGTCAAATACCAAGATATGATTGCGGCCCTGCCCGAAGCCACGCACGAGGCAATTGTAGGGGTCGGTAAACAGTGGTCGGGAGCGCGAGCACTTGAGGCGCTGCTGACTGTGGCGGGTGAGCTTAGGGGGCCTCCGCTCCAGCTCGACACCGGGCAGCTGCTGAAGATCGCGAAGAGAGGGGGAGTAACAGCGGTAGAGGCAGTGCACGCCTGGCGCAATGCGCTCACCGGGGCCCCCTTGAACCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAACATTGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCACATGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAACATCGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCCACGACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGACTCACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAACGGAGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAATAACAATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCCACGACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGGCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAACATTGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGATCAAGTCGTGGCCATTGCAAATAATAACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCCATGATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAACGGTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGTTTGACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAACGGAGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCCATGATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAATATTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGCCTGACACCCGAACAGGTGGTCGCCATTGCTAGCAACGGGGGAGGACGGCCAGCCTTGGAGTCCATCGTAGCCCAATTGTCCAGGCCCGATCCCGCGTTGGCTGCGTTAACGAATGACCATCTGGTGGCGTTGGCATGTCTTGGTGGACGACCCGCGCTCGATGCAGTCAAAAAGGGTCTGCCTCATGCTCCCGCATTGATCAAAAGAACCAACCGGCGGATTCCCGAGAGAACTTCCCATCGAGTCGCGGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT > CCR5 R549 (+28) (SEQ ID NO: 287)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS > CCR5R549 (+28) (SEQ ID NO: 288)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTATGGTGGACTTGAGGACACTCGGTTATTCGCAACAGCAACAGGAGAAAATCAAGCCTAAGGTCAGGAGCACCGTCGCGCAACACCACGAGGCGCTTGTGGGGCATGGCTTCACTCATGCGCATATTGTCGCGCTTTCACAGCACCCTGCGGCGCTTGGGACGGTGGCTGTCAAATACCAAGATATGATTGCGGCCCTGCCCGAAGCCACGCACGAGGCAATTGTAGGGGTCGGTAAACAGTGGTCGGGAGCGCGAGCACTTGAGGCGCTGCTGACTGTGGCGGGTGAGCTTAGGGGGCCTCCGCTCCAGCTCGACACCGGGCAGCTGCTGAAGATCGCGAAGAGAGGGGGAGTAACAGCGGTAGAGGCAGTGCACGCCTGGCGCAATGCGCTCACCGGGGCCCCCTTGAACCTGACCCCAGACCAGGTAGTCGCAATCGCCAACAATAACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAATATCGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAATGGGGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAATATTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGACTCACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAACGGAGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAATAACAATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAATATTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGGCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAACGGAGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAATAACAATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAACGGTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGTTTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAACATTGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTACTGTGCCAGGATCATGGCCTGACACCCGAACAGGTGGTCGCCATTGCTAGCAACGGGGGAGGACGGCCAGCCTTGGAGTCCATCGTAGCCCAATTGTCCAGGCCCGATCCCGCGTTGGCTGCGTTAACGAATGACCATCTGGTGGCGTTGGCATGTCTTGGTGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGTGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT > CCR5R549 (+63) (SEQ ID NO: 289)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS > CCR5 R549 (+63) (SEQ ID NO: 290)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTATGGTGGACTTGAGGACACTCGGTTATTCGCAACAGCAACAGGAGAAAATCAAGCCTAAGGTCAGGAGCACCGTCGCGCAACACCACGAGGCGCTTGTGGGGCATGGCTTCACTCATGCGCATATTGTCGCGCTTTCACAGCACCCTGCGGCGCTTGGGACGGTGGCTGTCAAATACCAAGATATGATTGCGGCCCTGCCCGAAGCCACGCACGAGGCAATTGTAGGGGTCGGTAAACAGTGGTCGGGAGCGCGAGCACTTGAGGCGCTGCTGACTGTGGCGGGTGAGCTTAGGGGGCCTCCGCTCCAGCTCGACACCGGGCAGCTGCTGAAGATCGCGAAGAGAGGGGGAGTAACAGCGGTAGAGGCAGTGCACGCCTGGCGCAATGCGCTCACCGGGGCCCCCTTGAACCTGACCCCAGACCAGGTAGTCGCAATCGCCAACAATAACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAATATCGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAATGGGGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAATATTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGACTCACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAACGGAGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAATAACAATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAATATTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGGCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAACGGAGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAATAACAATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAACGGTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGTTTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAACATTGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTACTGTGCCAGGATCATGGCCTGACACCCGAACAGGTGGTCGCCATTGCTAGCAACGGGGGAGGACGGCCAGCCTTGGAGTCCATCGTAGCCCAATTGTCCAGGCCCGATCCCGCGTTGGCTGCGTTAACGAATGACCATCTGGTGGCGTTGGCATGTCTTGGTGGACGACCCGCGCTCGATGCAGTCAAAAAGGGTCTGCCTCATGCTCCCGCATTGATCAAAAGAACCAACCGGCGGATTCCCGAGAGAACTTCCCATCGAGTCGCGGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT > CCR5R551 (+28) (SEQ ID NO: 291)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQTLETVQRLLPVLCQAHGLTPAQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS > CCR5 R551(+28) (SEQ ID NO: 292)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTATGGTGGACTTGAGGACACTCGGTTATTCGCAACAGCAACAGGAGAAAATCAAGCCTAAGGTCAGGAGCACCGTCGCGCAACACCACGAGGCGCTTGTGGGGCATGGCTTCACTCATGCGCATATTGTCGCGCTTTCACAGCACCCTGCGGCGCTTGGGACGGTGGCTGTCAAATACCAAGATATGATTGCGGCCCTGCCCGAAGCCACGCACGAGGCAATTGTAGGGGTCGGTAAACAGTGGTCGGGAGCGCGAGCACTTGAGGCGCTGCTGACTGTGGCGGGTGAGCTTAGGGGGCCTCCGCTCCAGCTCGACACCGGGCAGCTGCTGAAGATCGCGAAGAGAGGGGGAGTAACAGCGGTAGAGGCAGTGCACGCCTGGCGCAATGCGCTCACCGGGGCCCCCTTGAACCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAATGGCGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGATCAAGTCGTGGCCATTGCAAATAATAACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAACATCGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAACGGTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGACTCACCCCAGACCAGGTAGTCGCAATCGCGTCGAACATTGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCACATGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAATGGGGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCAACAACAACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGGCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAACATTGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCACATGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAATGGGGGAGGGAAACAAACATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCAACAACAACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGCCTGACACCCGAACAGGTGGTCGCCATTGCTAGCAACGGGGGAGGACGGCCAGCCTTGGAGTCCATCGTAGCCCAATTGTCCAGGCCCGATCCCGCGTTGGCTGCGTTAACGAATGACCATCTGGTGGCGTTGGCATGTCTTGGTGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT > CCR5R551 (+63) (SEQ ID NO: 293)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS > CCR5R551 (+63) (SEQ ID NO: 294)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTATGGTGGACTTGAGGACACTCGGTTATTCGCAACAGCAACAGGAGAAAATCAAGCCTAAGGTCAGGAGCACCGTCGCGCAACACCACGAGGCGCTTGTGGGGCATGGCTTCACTCATGCGCATATTGTCGCGCTTTCACAGCACCCTGCGGCGCTTGGGACGGTGGCTGTCAAATACCAAGATATGATTGCGGCCCTGCCCGAAGCCACGCACGAGGCAATTGTAGGGGTCGGTAAACAGTGGTCGGGAGCGCGAGCACTTGAGGCGCTGCTGACTGTGGCGGGTGAGCTTAGGGGGCCTCCGCTCCAGCTCGACACCGGGCAGCTGCTGAAGATCGCGAAGAGAGGGGGAGTAACAGCGGTAGAGGCAGTGCACGCCTGGCGCAATGCGCTCACCGGGGCCCCCTTGAACCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAATGGCGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGATCAAGTCGTGGCCATTGCAAATAATAACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAACATCGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAACGGTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGACTCACCCCAGACCAGGTAGTCGCAATCGCGTCGAACATTGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCACATGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAATGGGGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCAACAACAACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGGCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAACATTGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCACATGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAATGGGGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCAACAACAACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGCCTGACACCCGAACAGGTGGTCGCCATTGCTAGCAACGGGGGAGGACGGCCAGCCTTGGAGTCCATCGTAGCCCAATTGTCCAGGCCCGATCCCGCGTTGGCTGCGTTAACGAATGACCATCTGGTGGCGTTGGCATGTCTTGGTGGACGACCCGCGCTCGATGCAGTCAAAAAGGGTCTGCCTCATGCTCCCGCATTGATCAAAAGAACCAACCGGCGGATTCCCGAGAGAACTTCCCATCGAGTCGCGGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT > CCR5R557 (+28) (SEQ ID NO: 295)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS > CCR5R557 (+28) (SEQ ID NO: 296)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTATGGTGGACTTGAGGACACTCGGTTATTCGCAACAGCAACAGGAGAAAATCAAGCCTAAGGTCAGGAGCACCGTCGCGCAACACCACGAGGCGCTTGTGGGGCATGGCTTCACTCATGCGCATATTGTCGCGCTTTCACAGCACCCTGCGGCGCTTGGGACGGTGGCTGTCAAATACCAAGATATGATTGCGGCCCTGCCCGAAGCCACGCACGAGGCAATTGTAGGGGTCGGTAAACAGTGGTCGGGAGCGCGAGCACTTGAGGCGCTGCTGACTGTGGCGGGTGAGCTTAGGGGGCCTCCGCTCCAGCTCGACACCGGGCAGCTGCTGAAGATCGCGAAGAGAGGGGGAGTAACAGCGGTAGAGGCAGTGCACGCCTGGCGCAATGCGCTCACCGGGGCCCCCTTGAACCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAACGGAGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAATGGGGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCCACGACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGACTCACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAATATCGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAATAACAATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAATATTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGGCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAACATTGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAACGGAGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAATGGGCGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCAACAACAACGGCGCTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGTTTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAACATTGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAACGGAGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAACATCGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCCACGACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGCCTGACACCCGAACAGGTGGTCGCCATTGCTAGCAACGGGGGAGGACGGCCAGCCTTGGAGTCCATCGTAGCCCAATTGTCCAGGCCCGATCCCGCGTTGGCTGCGTTAACGAATGACCATCTGGTGGCGTTGGCATGTCTTGGTGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT > CCR5R557 (+63) (SEQ ID NO: 297)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS > CCR5 R557 (+63) (SEQ ID NO: 298)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTATGGTGGACTTGAGGACACTCGGTTATTCGCAACAGCAACAGGAGAAAATCAAGCCTAAGGTCAGGAGCACCGTCGCGCAACACCACGAGGCGCTTGTGGGGCATGGCTTCACTCATGCGCATATTGTCGCGCTTTCACAGCACCCTGCGGCGCTTGGGACGGTGGCTGTCAAATACCAAGATATGATTGCGGCCCTGCCCGAAGCCACGCACGAGGCAATTGTAGGGGTCGGTAAACAGTGGTCGGGAGCGCGAGCACTTGAGGCGCTGCTGACTGTGGCGGGTGAGCTTAGGGGGCCTCCGCTCCAGCTCGACACCGGGCAGCTGCTGAAGATCGCGAAGAGAGGGGGAGTAACAGCGGTAGAGGCAGTGCACGCCTGGCGCAATGCGCTCACCGGGGCCCCCTTGAACCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAACGGAGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAATGGGGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCCACGACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGACTCACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAATATCGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAATAACAATGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAATATTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGGCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAACATTGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAACGGAGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAATGGGGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCAACAACAACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGTTTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAACATTGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAACGGAGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAACATCGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCCACGACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGCCTGACACCCGAACAGGTGGTCGCCATTGCTAGCAACGGGGGAGGACGGCCAGCCTTGGAGTCCATCGTAGCCCAATTGTCCAGGCCCGATCCCGCGTTGGCTGCGTTAACGAATGACCATCTGGTGGCGTTGGCATGTCTTGGTGGACGACCCGCGCTCGATGCAGTCAAAAAGGGTCTGCCTCATGCTCCCGCATTGATCAAAAGAACCAACCGGCGGATTCCCGAGAGAACTTCCCATCGAGTCGCGGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT > CCR5 R560 (+28) (SEQ ID NO: 299)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS > CCR5 R560 (+28) (SEQ ID NO: 300)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTATGGTGGACTTGAGGACACTCGGTTATTCGCAACAGCAACAGGAGAAAATCAAGCCTAAGGTCAGGAGCACCGTCGCGCAACACCACGAGGCGCTTGTGGGGCATGGCTTCACTCATGCGCATATTGTCGCGCTTTCACAGCACCCTGCGGCGCTTGGGACGGTGGCTGTCAAATACCAAGATATGATTGCGGCCCTGCCCGAAGCCACGCACGAGGCAATTGTAGGGGTCGGTAAACAGTGGTCGGGAGCGCGAGCACTTGAGGCGCTGCTGACTGTGGCGGGTGAGCTTAGGGGGCCTCCGCTCCAGCTCGACACCGGGCAGCTGCTGAAGATCGCGAAGAGAGGGGGAGTAACAGCGGTAGAGGCAGTGCACGCCTGGCGCAATGCGCTCACCGGGGCCCCCTTGAACCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAATGGCGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCACATGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAATGGGGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAACGGTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGACTCACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCACATGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAACATCGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCAACAACAACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGGCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAACATTGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAATATCGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAATGGGGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAACGGTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGTTTGACCCCAGACCAGGTAGTCGCAATCGCCAACAATAACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACGCCTGCACAAGTGGTCGCCATCGCCTCCAATATTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGCCTGACACCCGAACAGGTGGTCGCCATTGCTAGCAACGGGGGAGGACGGCCAGCCTTGGAGTCCATCGTAGCCCAATTGTCCAGGCCCGATCCCGCGTTGGCTGCGTTAACGAATGACCATCTGGTGGCGTTGGCATGTCTTGGTGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCT > CCR5R560 (+63) (SEQ ID NO: 301)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS > CCR5R560 (+63) (SEQ ID NO: 302)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTATGGTGGACTTGAGGACACTCGGTTATTCGCAACAGCAACAGGAGAAAATCAAGCCTAAGGTCAGGAGCACCGTCGCGCAACACCACGAGGCGCTTGTGGGGCATGGCTTCACTCATGCGCATATTGTCGCGCTTTCACAGCACCCTGCGGCGCTTGGGACGGTGGCTGTCAAATACCAAGATATGATTGCGGCCCTGCCCGAAGCCACGCACGAGGCAATTGTAGGGGTCGGTAAACAGTGGTCGGGAGCGCGAGCACTTGAGGCGCTGCTGACTGTGGCGGGTGAGCTTAGGGGGCCTCCGCTCCAGCTCGACACCGGGCAGCTGCTGAAGATCGCGAAGAGAGGGGGAGTAACAGCGGTAGAGGCAGTGCACGCCTGGCGCAATGCGCTCACCGGGGCCCCCTTGAACCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAATGGCGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCACATGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAATGGGGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAACGGTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGACTCACCCCAGACCAGGTAGTCGCAATCGCGTCGCATGACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCACATGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAACATCGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCAACAACAACGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGGCTGACCCCAGACCAGGTAGTCGCAATCGCGTCGAACATTGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCAAATATCGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGAGCAATGGGGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAACGGTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGTTTGACCCCAGACCAGGTAGTCGCAATCGCCAACAATAACGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACGCCTGCACAAGTGGTCGCCATCGCCTCCAATATTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGCCTGACACCCGAACAGGTGGTCGCCATTGCTAGCAACGGGGGAGGACGGCCAGCCTTGGAGTCCATCGTAGCCCAATTGTCCAGGCCCGATCCCGCGTTGGCTGCGTTAACGAATGACCATCTGGTGGCGTTGGCATGTCTTGGTGGACGACCCGCGCTCGATGCAGTCAAAAAGGGTCTGCCTCATGCTCCCGCATTGATCAAAAGAACCAACCGGCGGATTCCCGAGAGAACTTCCCATCGAGTCGCGGGATCCCAGCTGGTGAAGAGCGAGCTGGAGGAGAAGAAGTCCGAGCTGCGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCAGGAACAGCACCCAGGACCGCATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGAAAGCACCTGGGCGGAAGCAGAAAGCCTGACGGCGCCATCTATACAGTGGGCAGCCCCATCGATTACGGCGTGATCGTGGACACAAAGGCCTACAGCGGCGGCTACAATCTGCCTATCGGCCAGGCCGACGAGATGCAGAGATACGTGGAGGAGAACCAGACCCGGAATAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCTAGCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGAGCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAATGGCGCCGTGCTGAGCGTGGAGGAGCTGCTGATCGGCGGCGAGATGATCAAAGCCGGCACCCTGACACTGGAGGAGGTGCGGCGCAAGTTCAACAACGGCGAGATCAACTTCAGATCTCCR5 Donor Sequence: (SEQ ID NO: 176)5′AGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCAGAATTAACCCTCACTAAAGGGACTAGTCCTGCAGGTTTAAACGAATTCGCCCTTGATACTTATTAACCATACCTTGGAGGGGAAATCACACATGAAAAGTGTCATTTCTTTACTAATCATATTCATGTCTTTTCTCCCCATAGCAAGACAAAGACCTGTTTTAAACACATTTACAACCTATATGTTGCCTTGTACTAGGTAAAAAGTTGTACATTTCTGAAATAATTTTGGTATTTCTGTTCAGATCACTAAACTCAAGAATCAGCAATTCTCTGAGGCTTTCTTTTAAATATACATAAGGAACTTTCGGAGTGAAGGGAGAGTTTGTCAATAACTTGATGCATGTGAAGGGGAGATAAAAAGGTTGCTATTTTTCATCAACATATTTTGATTTGGCTTTCTATAATTGATGGGCTTAAAAGATCTAATCTACTTTAAACAGATGCCAAATAAATGGATGAATCTTAGACCCTCTATAACAGTAACTTCCTTTTAAAAAAGACCTCTCCCACCCCACCCCCAGCCCAGGCTGTGTATGAAAACTAAGCCATGTGCACAACTCTGACTGGGTCACCAGCCCACTTGAGTCCGTGTCACAAGCCCACAGATATTTCCTGCTCCCCAGTGGATCGGGTGTAAACTGAGCTTGCTCGCTCGGGAGCCTCTTGCTGGAAAATAGAACAGCATTTGCAGAAGCGTTTGGCAATGTGCTTTTGGAAGAAGACTAAGAGGTAGTTTCTGAACTTCTCCCCGACAAAGGCATAGATGATGGGGTTGATGCAGCAGTGCGTCATCCCAAGAGTCTCTGTCACCTGCATAGCTTGGTCCAACCTGTTAGAGCTACTGCAATTATTCAGGCCAAAGAATTCCTGGAAGGTGTTCAGGAGAAGGACAATGTTGTAGGGAGCCCAGAAGAGAAAATAAACAATCATGATGGTGAAGATAAGCCTCACAGCCCTGTGCCTCTTCTTCTCATTTCGACACCGAAGCAGAGTTTTTAGGATTCCCGAGTAGCAGATGACCATGACAAGCAGCGGCAGGACCAGCCCCAAGATGACTATCTTTAATGTCTGGAAATTCTTCCAGAATTGATACTGACTGTATGGAAAATGAGAGCTGCAGGTGTAATGAAGACCTTCTTTTTGAGATCTGGTAAAGATGATTCCTGGGAGAGACGCAAACACAGCCACCACCCAAGTGATCACACTTGTCACCACCCCAAAGGTGACCGTCCTGGCTTTTAAAGCAAACACAGCATGGACGACAGCCAGGTACCTATCGATTGTCAGGAGGATGATGAAGAAGATTCCAGAGAAGAAGCCTATAAAATAGAGCCCTGTCAAGAGTTGACACATTGTATTTCCAAAGTCCCACTGGGCGGCAGCATAGTGAGCCCAGAAGGGGACAGTAAGAAGGAAAAACAGGTCAGAGATGGCCAGGTTGAGCAGGTAGATGTCAGTCATGCTCTTCAGCCTTTTGCAGTTTTCTAGACGAGGCATCCAGTCCAGACGCCATCAGGGCATACTCACTGATCTAGATGAGGATGACCAGCATGTTGCCCACAAAACCAAAGATGAACACCAGTGAGTAGAGCGGAGGCAGGAGGCGGGCTGCGATTTGCTTCACATTGATTTTTTGGCAGGGCTCCGATGTATAATAATTGATGTCATAGATTGGACTTGACACTTGATAATCCATCTTGTTCCACCCTGTGCATAAATAAAAAGTGATCTTTTATAAAGTCCTAGAATGTATTTAGTTGCCCTCCATGAATGCAAACTGTTTTATACATCAATAGGTTTTTAATTGCCTACATAGATGTCTACATTGAATTAACTCTCTTTTTGGCCAAGCAATGAAGTTTTGTAGTGAAGGGAAGGTTTGCTGCTAGCTTCCCTGTCCACTAGATGGAGAGCTTGGCTCTGTTGGGGGAATTCATGAAAGCACCATCTCACCAAATAAAATCTTGTGCTCTATAGCACCATGGAGTGAATGAAGCTTTGACAACAATTAAGGGCGAATTCGCGGCCGCTAAATTCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTATACGTACGGCAGTTTAAGGTTTACACCTATAAAAGAGAGAGCCGTTATCGTCTGTTTGTGGATGTACAGAGTGATATTATTGACACGCCGGGGCGACGGATGGTGATCCCCCTGGCCAGTGCACGTCTGCTGTCAGATAAAGTCTCCCGTGAACTTTACCCGGTGGTGCATATCGGGGATGAAAGCTGGCGCATGATGACCACCGATATGGCCAGTGTGCCGGTCTCCGTTATCGGGGAAGAAGTGGCTGATCTCAGCCACCGCGAAAATGACATCAAAAACGCCATTAACCTGATGTTCTGGGGAATATAAATGTCAGGCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTCACGTAGAAAGCCAGTCCGCAGAAACGGTGCTGACCCCGGATGAATGTCAGCTACTGGGCTATCTGGACAAGGGAAAACGCAAGCGCAAAGAGAAAGCAGGTAGCTTGCAGTGGGCTTACATGGCGATAGCTAGACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAATTGCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGGATGGCTTTCTTGCCGCCAAGGATCTGATGGCGCAGGGGATCAAGCTCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGAGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAATTATTAACGCTTACAATTTCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAG3′

3. TALE Constructs and Protein Sequences Used Gene Activation Studies

Complete TALE Construct Sequence, with Coding Sequence Underlined (SEQID NO:303):

TAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCTTAAGCTGATCCACTAGTCCAGTGTGGTGGAATTCGCTAGCGCCACCATGGCCCCCAAGAAGAAGAGGAAGGTGGGAATCGATGGGGTACCCGCCGCTGTGGATCTACGCACGCTCGGCTACAGCCAGCAGCAACAGGAGAAGATCAAACCGAAGGTTCGTTCGACAGTGGCGCAGCACCACGAGGCACTGGTCGGCCATGGGTTTACACACGCGCACATCGTTGCGCTCAGCCAACACCCGGCAGCGTTAGGGACCGTCGCTGTCAAGTATCAGGACATGATCGCAGCGTTGCCAGAGGCGACACACGAAGCGATCGTTGGCGTCGGCAAACAGTGGTCCGGCGCACGCGCCCTGGAGGCCTTGCTCACGGTGGCGGGAGAGTTGAGAGGTCCACCGTTACAGTTGGACACAGGCCAACTTCTCAAGATTGCAAAACGTGGCGGCGTGACCGCAGTGGAGGCAGTGCATGCATGGCGCAATGCACTGACGGGGGCCCCCCTGAACCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGCACAGGTGGTGGCCATCGCCAGCAATATTGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGCGCCAGGCCCATGGCCTGACCCCGGAGCAGGTCGTGGCCATCGCCAGCAATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGCACAGGTGGTGGCCATCGCCAGCAATATTGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTCGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGACCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGACCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATGGCGGCGGCAGGCCGGCGCTGGAGAGCATTGTTGCCCAGTTATCTCGCCCTGATCCGGCGTTGGCCGCGTTAACCAACGACCACCTCGTCGCCTTGGCCTGCCTCGGCGGACGTCCTGCGCTGGATGCAGTGAAAAAGGGATTGCCGCACGCGCCGGCCTTGATCAAAAGAACCAATCGCCGTATTCCCGAACGCACATCCCATCGCGTTGCCGACCACGCGCAAGTGGTTCGCGTGCTGGGTTTTTTCCAGTGCCACTCCCACCCAGCGCAAGCATTTGATGACGCCATGACGCAGTTCGGGATGAGCAGGCACGGGTTGTTACAGCTCTTTCGCAGAGTGGGCGTCACCGAACTCGAAGCCCGCAGTGGAACGCTCCCCCCAGCCTCGCAGCGTTGGGACCGTATCCTCCAGGCATCAGGGATGAAAAGGGCCAAACCGTCCCCTACTTCAACTCAAACGCCGGACCAGGCGTCTTTGCATGCATTCGCCGATTCGCTGGAGCGTGACCTTGATGCGCCCAGCCCAACGCACGAGGGAGATCAGAGGCGGGCAAGCAGCCGTAAACGGTCCCGATCGGATCGTGCTGTCACCGGTCCCTCCGCACAGCAATCGTTCGAGGTGCGCGCTCCCGAACAGCGCGATGCGCTGCATTTGCCCCTCAGTTGGAGGGTAAAACGCCCGCGTACCAGTATCGGGGGCGGCCTCCCGGATCCTGGTACGCCCACGGCTGCCGACCTGGCAGCGTCCAGCACCGTGATGCGGGAACAAGATGAGGACCCCTTCGCAGGGGCAGCGGATGATTTCCCGGCATTCAACGAAGAGGAGCTCGCATGGTTGATGGAGCTATTGCCTCAGGACCGCGGCCGCGCCCCCCCGACCGATGTCAGCCTGGGGGACGAGCTCCACTTAGACGGCGAGGACGTGGCGATGGCGCATGCCGACGCGCTAGACGATTTCGATCTGGACATGTTGGGGGACGGGGATTCCCCGGGTCCGGGATTTACCCCCCACGACTCCGCCCCCTACGGCGCTCTGGATATGGCCGACTTCGAGTTTGAGCAGATGTTTACCGATGCCCTTGGAATTGACGAGTACGGTGGCGGCCGCGACTACAAGGACGACGATGACAAGTAAGCTTCTCGAGTCTAGCTAGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGCATCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGGGGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCGACGGATCGGGAGATCTCCCGATCCCCTATGGTCGACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAAT

Complete Protein and Coding Sequence for Each TALE Used in GeneActivation Studies:

To regenerate the sequence of each expression construct, replace theunderlined region of the above construct with each CDS shown below.

Note that the NT-L +95 protein includes a nuclear localization sequence(NLS) from SV40, while nuclear import for NT-L +278 relies on endogenouslocalization sequences present in the TALE C-terminal flanking region³.

>NT-L +278 VP16 (SEQ ID NO: 304)MVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPEQVVAIASNKGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNKGGRPALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQLFRAVGVTELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFADSLERDLDAPSPTHEGDQRRASSRKRSRSDRAVTGPSAQQSFEVRAPEQRDALHLPLSWRVKRPRTSIGGGLPDPGTPTAADLAASSTVMREQDEDPFAGAADDFPAFNEEELAWLMELLPQDRGRAPPTDVSLGDELHLDGEDVAMAHADALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGGGRDYKDDDDK >NT-L +278 VP16 (SEQ ID NO: 305)ATGGTGGATCTACGCACGCTCGGCTACAGCCAGCAGCAACAGGAGAAGATCAAACCGAAGGTTCGTTCGACAGTGGCGCAGCACCACGAGGCACTGGTCGGCCATGGGTTTACACACGCGCACATCGTTGCGCTCAGCCAACACCCGGCAGCGTTAGGGACCGTCGCTGTCAAGTATCAGGACATGATCGCAGCGTTGCCAGAGGCGACACACGAAGCGATCGTTGGCGTCGGCAAACAGTGGTCCGGCGCACGCGCCCTGGAGGCCTTGCTCACGGTGGCGGGAGAGTTGAGAGGTCCACCGTTACAGTTGGACACAGGCCAACTTCTCAAGATTGCAAAACGTGGCGGCGTGACCGCAGTGGAGGCAGTGCATGCATGGCGCAATGCACTGACGGGGGCCCCCCTGAACCTTACTCCCGAACAAGTAGTAGCGATAGCCAGTAATAACGGAGGTAAACAAGCCTTGGAGACGGTCCAAAGGTTGCTCCCGGTCTTGTGTCAGGCACATGGGCTGACGCCTCAACAGGTCGTCGCGATAGCGTCTAATAATGGAGGAAAGCAAGCTCTGGAAACCGTCCAGCGACTCCTTCCGGTTCTGTGCCAGGCTCATGGTCTGACTCCGCAGCAAGTCGTTGCTATAGCGTCCAACATCGGAGGCAAACAGGCCCTGGAGACCGTGCAGCGGTTGTTGCCTGTGCTTTGCCAAGCCCACGGGCTTACGCCTGAGCAAGTGGTGGCGATTGCCAGTAACAACGGCGGCAAACAAGCCCTTGAGACTGTGCAGAGGCTCTTGCCGGTACTCTGCCAAGCACACGGCTTGACCCCCGAGCAGGTTGTAGCCATAGCTAGTCACGACGGGGGTAAGCAAGCGTTGGAAACGGTGCAAGCACTTCTCCCCGTTCTCTGTCAAGCGCATGGACTTACCCCGGAACAGGTGGTCGCCATTGCAAGCCATGATGGAGGAAAGCAGGCGCTCGAAACAGTCCAGGCACTTTTGCCCGTACTTTGTCAAGCTCACGGTCTCACCCCGGAACAGGTGGTAGCCATTGCATCTAACATCGGAGGTAAGCAAGCATTGGAAACGGTTCAGGCCCTGTTGCCTGTACTTTGCCAGGCGCACGGTCTGACACCTGAGCAGGTTGTCGCCATCGCTAGCAACGGAGGTGGGAAACAGGCACTTGAAACTGTGCAGAGGCTTCTGCCGGTGCTGTGCCAAGCGCATGGCCTTACACCCGAGCAAGTAGTGGCTATTGCGAGTCATGATGGAGGCAAGCAAGCGCTGGAGACTGTCCAACGACTTCTTCCGGTCTTGTGTCAGGCACATGGATTGACCCCTCAACAAGTCGTGGCGATAGCTAGCAACGGCGGTGGAAAACAGGCCCTCGAAACCGTCCAGCGACTGCTCCCCGTACTGTGTCAAGCCCATGGACTTACCCCAGAACAAGTTGTGGCGATTGCCTCTAACAATGGTGGGAAGCAAGCTCTTGAGACGGTGCAGGCGTTGTTGCCCGTGCTTTGTCAAGCTCACGGGCTCACGCCAGAGCAAGTGGTCGCTATCGCGAGTAATAAAGGGGGCAAACAAGCCTTGGAGACAGTGCAAAGGCTCCTGCCAGTGCTCTGCCAGGCTCATGGTTTGACACCCGAACAGGTAGTTGCAATAGCGAGTCATGATGGCGGAAAGCAAGCTCTTGAAACTGTGCAGCGGCTGTTGCCTGTACTGTGTCAAGCCCACGGGCTGACACCGGAACAAGTTGTAGCGATCGCTAGCCACGATGGCGGGAAACAAGCTCTGGAAACGGTACAGAGACTCCTCCCAGTGCTTTGTCAGGCACACGGCCTCACGCCAGAGCAGGTTGTCGCCATCGCGTCAAACAATGGTGGAAAGCAGGCCCTGGAGACAGTCCAACGGTTGCTGCCGGTCCTTTGCCAGGCTCACGGGTTGACCCCCCAGCAGGTCGTGGCCATTGCCTCAAACAAGGGCGGTAGGCCAGCATTGGAGACGGTGCAGAGGCTTCTGCCTGTGCTCTGCCAAGCGCATGGACTCACCCCCGAGCAAGTGGTTGCTATCGCAAGTAACAACGGAGGGAAACAAGCGCTCGAAACCGTGCAAAGGTTGCTCCCCGTTCTCTGTCAGGCGCACGGTCTTACGCCACAACAGGTGGTGGCGATTGCATCTAATGGAGGCGGACGCCCTGCCTTGGAGAGCATTGTGGCCCAGCTGTCCAGGCCGGACCCTGCCCTGGCCGCGTTAACCAACGACCACCTCGTCGCCTTGGCCTGCCTCGGCGGACGTCCTGCGCTGGATGCAGTGAAAAAGGGATTGCCGCACGCGCCGGCCTTGATCAAAAGAACCAATCGCCGTATTCCCGAACGCACATCCCATCGCGTTGCCGACCACGCGCAAGTGGTTCGCGTGCTGGGTTTTTTCCAGTGCCACTCCCACCCAGCGCAAGCATTTGATGACGCCATGACGCAGTTCGGGATGAGCAGGCACGGGTTGTTACAGCTCTTTCGCAGAGTGGGCGTCACCGAACTCGAAGCCCGCAGTGGAACGCTCCCCCCAGCCTCGCAGCGTTGGGACCGTATCCTCCAGGCATCAGGGATGAAAAGGGCCAAACCGTCCCCTACTTCAACTCAAACGCCGGACCAGGCGTCTTTGCATGCATTCGCCGATTCGCTGGAGCGTGACCTTGATGCGCCCAGCCCAACGCACGAGGGAGATCAGAGGCGGGCAAGCAGCCGTAAACGGTCCCGATCGGATCGTGCTGTCACCGGTCCCTCCGCACAGCAATCGTTCGAGGTGCGCGCTCCCGAACAGCGCGATGCGCTGCATTTGCCCCTCAGTTGGAGGGTAAAACGCCCGCGTACCAGTATCGGGGGCGGCCTCCCGGATCCTGGTACGCCCACGGCTGCCGACCTGGCAGCGTCCAGCACCGTGATGCGGGAACAAGATGAGGACCCCTTCGCAGGGGCAGCGGATGATTTCCCGGCATTCAACGAAGAGGAGCTCGCATGGTTGATGGAGCTATTGCCTCAGGACCGCGGCCGCGCCCCCCCGACCGATGTCAGCCTGGGGGACGAGCTCCACTTAGACGGCGAGGACGTGGCGATGGCGCATGCCGACGCGCTAGACGATTTCGATCTGGACATGTTGGGGGACGGGGATTCCCCGGGTCCGGGATTTACCCCCCACGACTCCGCCCCCTACGGCGCTCTGGATATGGCCGACTTCGAGTTTGAGCAGATGTTTACCGATGCCCTTGGAATTGACGAGTACGGTGGCGGCCGCGACTACAAGGACGACGATGACAAG >NT-L +95 VP16 (SEQ ID NO: 306)MAPKKKRKVGIDGVPAAVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGOLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPEQVVAIASNKGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNKGGRPALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSGSRGRAPPTDVSLGDELHLDGEDVAMAHADALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGGGRDYKDDDDK >NT-L +95 VP16(SEQ ID NO: 307)ATGGCCCCCAAGAAGAAGAGGAAGGTGGGAATCGATGGGGTACCCGCCGCTGTGGATCTACGCACGCTCGGCTACAGCCAGCAGCAACAGGAGAAGATCAAACCGAAGGTTCGTTCGACAGTGGCGCAGCACCACGAGGCACTGGTCGGCCATGGGTTTACACACGCGCACATCGTTGCGCTCAGCCAACACCCGGCAGCGTTAGGGACCGTCGCTGTCAAGTATCAGGACATGATCGCAGCGTTGCCAGAGGCGACACACGAAGCGATCGTTGGCGTCGGCAAACAGTGGTCCGGCGCACGCGCCCTGGAGGCCTTGCTCACGGTGGCGGGAGAGTTGAGAGGTCCACCGTTACAGTTGGACACAGGCCAACTTCTCAAGATTGCAAAACGTGGCGGCGTGACCGCAGTGGAGGCAGTGCATGCATGGCGCAATGCACTGACGGGGGCCCCCCTGAACCTTACTCCCGAACAAGTAGTAGCGATAGCCAGTAATAACGGAGGTAAACAAGCCTTGGAGACGGTCCAAAGGTTGCTCCCGGTCTTGTGTCAGGCACATGGGCTGACGCCTCAACAGGTCGTCGCGATAGCGTCTAATAATGGAGGAAAGCAAGCTCTGGAAACCGTCCAGCGACTCCTTCCGGTTCTGTGCCAGGCTCATGGTCTGACTCCGCAGCAAGTCGTTGCTATAGCGTCCAACATCGGAGGCAAACAGGCCCTGGAGACCGTGCAGCGGTTGTTGCCTGTGCTTTGCCAAGCCCACGGGCTTACGCCTGAGCAAGTGGTGGCGATTGCCAGTAACAACGGCGGCAAACAAGCCCTTGAGACTGTGCAGAGGCTCTTGCCGGTACTCTGCCAAGCACACGGCTTGACCCCCGAGCAGGTTGTAGCCATAGCTAGTCACGACGGGGGTAAGCAAGCGTTGGAAACGGTGCAAGCACTTCTCCCCGTTCTCTGTCAAGCGCATGGACTTACCCCGGAACAGGTGGTCGCCATTGCAAGCCATGATGGAGGAAAGCAGGCGCTCGAAACAGTCCAGGCACTTTTGCCCGTACTTTGTCAAGCTCACGGTCTCACCCCGGAACAGGTGGTAGCCATTGCATCTAACATCGGAGGTAAGCAAGCATTGGAAACGGTTCAGGCCCTGTTGCCTGTACTTTGCCAGGCGCACGGTCTGACACCTGAGCAGGTTGTCGCCATCGCTAGCAACGGAGGTGGGAAACAGGCACTTGAAACTGTGCAGAGGCTTCTGCCGGTGCTGTGCCAAGCGCATGGCCTTACACCCGAGCAAGTAGTGGCTATTGCGAGTCATGATGGAGGCAAGCAAGCGCTGGAGACTGTCCAACGACTTCTTCCGGTCTTGTGTCAGGCACATGGATTGACCCCTCAACAAGTCGTGGCGATAGCTAGCAACGGCGGTGGAAAACAGGCCCTCGAAACCGTCCAGCGACTGCTCCCCGTACTGTGTCAAGCCCATGGACTTACCCCAGAACAAGTTGTGGCGATTGCCTCTAACAATGGTGGGAAGCAAGCTCTTGAGACGGTGCAGGCGTTGTTGCCCGTGCTTTGTCAAGCTCACGGGCTCACGCCAGAGCAAGTGGTCGCTATCGCGAGTAATAAAGGGGGCAAACAAGCCTTGGAGACAGTGCAAAGGCTCCTGCCAGTGCTCTGCCAGGCTCATGGTTTGACACCCGAACAGGTAGTTGCAATAGCGAGTCATGATGGCGGAAAGCAAGCTCTTGAAACTGTGCAGCGGCTGTTGCCTGTACTGTGTCAAGCCCACGGGCTGACACCGGAACAAGTTGTAGCGATCGCTAGCCACGATGGCGGGAAACAAGCTCTGGAAACGGTACAGAGACTCCTCCCAGTGCTTTGTCAGGCACACGGCCTCACGCCAGAGCAGGTTGTCGCCATCGCGTCAAACAATGGTGGAAAGCAGGCCCTGGAGACAGTCCAACGGTTGCTGCCGGTCCTTTGCCAGGCTCACGGGTTGACCCCCCAGCAGGTCGTGGCCATTGCCTCAAACAAGGGCGGTAGGCCAGCATTGGAGACGGTGCAGAGGCTTCTGCCTGTGCTCTGCCAAGCGCATGGACTCACCCCCGAGCAAGTGGTTGCTATCGCAAGTAACAACGGAGGGAAACAAGCGCTCGAAACCGTGCAAAGGTTGCTCCCCGTTCTCTGTCAGGCGCACGGTCTTACGCCACAACAGGTGGTGGCGATTGCATCTAATGGAGGCGGACGCCCTGCCTTGGAGAGCATTGTGGCCCAGCTGTCCAGGCCGGACCCTGCCCTGGCCGCGTTAACCAACGACCACCTCGTCGCCTTGGCCTGCCTCGGCGGACGTCCTGCGCTGGATGCAGTGAAAAAGGGATTGCCGCACGCGCCGGCCTTGATCAAAAGAACCAATCGCCGTATTCCCGAACGCACATCCCATCGCGTTGCCGACCACGCGCAAGTGGTTCGCGTGCTGGGTTTTTTCCAGTGCCACTCCCACCCAGCGCAAGCATTTGATGACGCCATGACGCAGTTCGGGATGAGCGGATCCCGCGGCCGCGCCCCCCCGACCGATGTCAGCCTGGGGGACGAGCTCCACTTAGACGGCGAGGACGTGGCGATGGCGCATGCCGACGCGCTAGACGATTTCGATCTGGACATGTTGGGGGACGGGGATTCCCCGGGTCCGGGATTTACCCCCCACGACTCCGCCCCCTACGGCGCTCTGGATATGGCCGACTTCGAGTTTGAGCAGATGTTTACCGATGCCCTTGGAATTGACGAGTACGGTGGCGGCCGCGACTACAAGGACGACGATGACAAG >TALE13 +278 VP16 (SEQ ID NO: 308)MAPKKKRKVGIDGVPAAVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLRQAHGLTPEQVVAIASNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQLFRRVGVTELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFADSLERDLDAPSPTHEGDQRRASSRKRSRSDRAVTGPSAQQSFEVRAPEQRDALHLPLSWRVKRPRTSIGGGLPDPGTPTAADLAASSTVMREQDEDPFAGAADDFPAFNEEELAWLMELLPQDRGRAPPTDVSLGDELHLDGEDVAMAHADALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGGGRDYKDDDDK >TALE13 +278 VP16(SEQ ID NO: 309)ATGGCCCCCAAGAAGAAGAGGAAGGTGGGAATCGATGGGGTACCCGCCGCTGTGGATCTACGCACGCTCGGCTACAGCCAGCAGCAACAGGAGAAGATCAAACCGAAGGTTCGTTCGACAGTGGCGCAGCACCACGAGGCACTGGTCGGCCATGGGTTTACACACGCGCACATCGTTGCGCTCAGCCAACACCCGGCAGCGTTAGGGACCGTCGCTGTCAAGTATCAGGACATGATCGCAGCGTTGCCAGAGGCGACACACGAAGCGATCGTTGGCGTCGGCAAACAGTGGTCCGGCGCACGCGCCCTGGAGGCCTTGCTCACGGTGGCGGGAGAGTTGAGAGGTCCACCGTTACAGTTGGACACAGGCCAACTTCTCAAGATTGCAAAACGTGGCGGCGTGACCGCAGTGGAGGCAGTGCATGCATGGCGCAATGCACTGACGGGGGCCCCCCTGAACCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGCACAGGTGGTGGCCATCGCCAGCAATATTGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGCGCCAGGCCCATGGCCTGACCCCGGAGCAGGTCGTGGCCATCGCCAGCAATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGCACAGGTGGTGGCCATCGCCAGCAATATTGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTCGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGACCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGACCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATGGCGGCGGCAGGCCGGCGCTGGAGAGCATTGTTGCCCAGTTATCTCGCCCTGATCCGGCGTTGGCCGCGTTAACCAACGACCACCTCGTCGCCTTGGCCTGCCTCGGCGGACGTCCTGCGCTGGATGCAGTGAAAAAGGGATTGCCGCACGCGCCGGCCTTGATCAAAAGAACCAATCGCCGTATTCCCGAACGCACATCCCATCGCGTTGCCGACCACGCGCAAGTGGTTCGCGTGCTGGGTTTTTTCCAGTGCCACTCCCACCCAGCGCAAGCATTTGATGACGCCATGACGCAGTTCGGGATGAGCAGGCACGGGTTGTTACAGCTCTTTCGCAGAGTGGGCGTCACCGAACTCGAAGCCCGCAGTGGAACGCTCCCCCCAGCCTCGCAGCGTTGGGACCGTATCCTCCAGGCATCAGGGATGAAAAGGGCCAAACCGTCCCCTACTTCAACTCAAACGCCGGACCAGGCGTCTTTGCATGCATTCGCCGATTCGCTGGAGCGTGACCTTGATGCGCCCAGCCCAACGCACGAGGGAGATCAGAGGCGGGCAAGCAGCCGTAAACGGTCCCGATCGGATCGTGCTGTCACCGGTCCCTCCGCACAGCAATCGTTCGAGGTGCGCGCTCCCGAACAGCGCGATGCGCTGCATTTGCCCCTCAGTTGGAGGGTAAAACGCCCGCGTACCAGTATCGGGGGCGGCCTCCCGGATCCTGGTACGCCCACGGCTGCCGACCTGGCAGCGTCCAGCACCGTGATGCGGGAACAAGATGAGGACCCCTTCGCAGGGGCAGCGGATGATTTCCCGGCATTCAACGAAGAGGAGCTCGCATGGTTGATGGAGCTATTGCCTCAGGACCGCGGCCGCGCCCCCCCGACCGATGTCAGCCTGGGGGACGAGCTCCACTTAGACGGCGAGGACGTGGCGATGGCGCATGCCGACGCGCTAGACGATTTCGATCTGGACATGTTGGGGGACGGGGATTCCCCGGGTCCGGGATTTACCCCCCACGACTCCGCCCCCTACGGCGCTCTGGATATGGCCGACTTCGAGTTTGAGCAGATGTTTACCGATGCCCTTGGAATTGACGAGTACGGTGGCGGCCGCGACTACAAGGACGACGATGACAAG >TALE13 +133 VP16 (SEQ ID NO: 310)MVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLRQAHGLTPEQVVAIASNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQLFRRVGVTELEARSGTLPPASQRWDRILQASGGSGHRGRAPPTDVSLGDELHLDGEDVAMAHADALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGGGRDYKDDDDK >TALE13 +133 VP16 (SEQ ID NO: 311)ATGGTGGATCTACGCACGCTCGGCTACAGCCAGCAGCAACAGGAGAAGATCAAACCGAAGGTTCGTTCGACAGTGGCGCAGCACCACGAGGCACTGGTCGGCCATGGGTTTACACACGCGCACATCGTTGCGCTCAGCCAACACCCGGCAGCGTTAGGGACCGTCGCTGTCAAGTATCAGGACATGATCGCAGCGTTGCCAGAGGCGACACACGAAGCGATCGTTGGCGTCGGCAAACAGTGGTCCGGCGCACGCGCCCTGGAGGCCTTGCTCACGGTGGCGGGAGAGTTGAGAGGTCCACCGTTACAGTTGGACACAGGCCAACTTCTCAAGATTGCAAAACGTGGCGGCGTGACCGCAGTGGAGGCAGTGCATGCATGGCGCAATGCACTGACGGGTGCCCCCCTGAACCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGCACAGGTGGTGGCCATCGCCAGCAATATTGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGCGCCAGGCCCATGGCCTGACCCCGGAGCAGGTCGTGGCCATCGCCAGCAATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGCACAGGTGGTGGCCATCGCCAGCAATATTGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTCGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGACCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGACCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATGGCGGCGGCAGGCCGGCGCTGGAGAGCATTGTTGCCCAGTTATCTCGCCCTGATCCGGCGTTGGCCGCGTTGACCAACGACCACCTCGTCGCCTTGGCCTGCCTCGGCGGACGTCCTGCGCTGGATGCAGTGAAAAAGGGATTGCCGCACGCGCCGGCCTTGATCAAAAGAACCAATCGCCGTATTCCCGAACGCACATCCCATCGCGTTGCCGACCACGCGCAAGTGGTTCGCGTGCTGGGTTTTTTCCAGTGCCACTCCCACCCAGCGCAAGCATTTGATGACGCCATGACGCAGTTCGGGATGAGCAGGCACGGGTTGTTACAGCTCTTTCGCAGAGTGGGCGTCACCGAACTCGAAGCCCGCAGTGGAACGCTCCCCCCAGCCTCGCAGCGTTGGGACCGTATCCTCCAGGCATCGGGGGGATCCGGCCACCGCGGCCGCGCCCCCCCGACCGATGTCAGCCTGGGGGACGAGCTCCACTTAGACGGCGAGGACGTGGCGATGGCGCATGCCGACGCGCTAGACGATTTCGATCTGGACATGTTGGGGGACGGGGATTCCCCGGGTCCGGGATTTACCCCCCACGACTCCGCCCCCTACGGCGCTCTGGATATGGCCGACTTCGAGTTTGAGCAGATGTTTACCGATGCCCTTGGAATTGACGAGTACGGTGGCGGCCGCGACTACAAGGACGACGATGACAAG >TALE13 +95 VP16(SEQ ID NO: 312)MVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLRQAHGLTPEQVVAIASNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSGSRGRAPPTDVSLGDELHLDGEDVAMAHADALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGGGRDYKDDDDK >TALE13 +95 VP16(SEQ ID NO: 313)ATGGTGGATCTACGCACGCTCGGCTACAGCCAGCAGCAACAGGAGAAGATCAAACCGAAGGTTCGTTCGACAGTGGCGCAGCACCACGAGGCACTGGTCGGCCATGGGTTTACACACGCGCACATCGTTGCGCTCAGCCAACACCCGGCAGCGTTAGGGACCGTCGCTGTCAAGTATCAGGACATGATCGCAGCGTTGCCAGAGGCGACACACGAAGCGATCGTTGGCGTCGGCAAACAGTGGTCCGGCGCACGCGCCCTGGAGGCCTTGCTCACGGTGGCGGGAGAGTTGAGAGGTCCACCGTTACAGTTGGACACAGGCCAACTTCTCAAGATTGCAAAACGTGGCGGCGTGACCGCAGTGGAGGCAGTGCATGCATGGCGCAATGCACTGACGGGGGCCCCCCTGAACCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGCACAGGTGGTGGCCATCGCCAGCAATATTGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGCGCCAGGCCCATGGCCTGACCCCGGAGCAGGTCGTGGCCATCGCCAGCAATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGCACAGGTGGTGGCCATCGCCAGCAATATTGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTCGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGACCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGACCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATGGCGGCGGCAGGCCGGCGCTGGAGAGCATTGTTGCCCAGTTATCTCGCCCTGATCCGGCGTTGGCCGCGTTAACCAACGACCACCTCGTCGCCTTGGCCTGCCTCGGCGGACGTCCTGCGCTGGATGCAGTGAAAAAGGGATTGCCGCACGCGCCGGCCTTGATCAAAAGAACCAATCGCCGTATTCCCGAACGCACATCCCATCGCGTTGCCGACCACGCGCAAGTGGTTCGCGTGCTGGGTTTTTTCCAGTGCCACTCCCACCCAGCGCAAGCATTTGATGACGCCATGACGCAGTTCGGGATGAGCGGATCCCGCGGCCGCGCCCCCCCGACCGATGTCAGCCTGGGGGACGAGCTCCACTTAGACGGCGAGGACGTGGCGATGGCGCATGCCGACGCGCTAGACGATTTCGATCTGGACATGTTGGGGGACGGGGATTCCCCGGGTCCGGGATTTACCCCCCACGACTCCGCCCCCTACGGCGCTCTGGATATGGCCGACTTCGAGTTTGAGCAGATGTTTACCGATGCCCTTGGAATTGACGAGTACGGTGGCGGCCGCGACTACAAGGACGACGATGACAAG >TALE13 +23 VP16 (SEQ ID NO: 314)MVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEATVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLRQAHGLTPEQVVAIASNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVAGSRGRAPPTDVSLGDELHLDGEDVAMAHADALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGGGRDYKDDDDK >TALE13 +23 VP16(SEQ ID NO: 315)ATGGTGGATCTACGCACGCTCGGCTACAGCCAGCAGCAACAGGAGAAGATCAAACCGAAGGTTCGTTCGACAGTGGCGCAGCACCACGAGGCACTGGTCGGCCATGGGTTTACACACGCGCACATCGTTGCGCTCAGCCAACACCCGGCAGCGTTAGGGACCGTCGCTGTCAAGTATCAGGACATGATCGCAGCGTTGCCAGAGGCGACACACGAAGCGATCGTTGGCGTCGGCAAACAGTGGTCCGGCGCACGCGCCCTGGAGGCCTTGCTCACGGTGGCGGGAGAGTTGAGAGGTCCACCGTTACAGTTGGACACAGGCCAACTTCTCAAGATTGCAAAACGTGGCGGCGTGACCGCAGTGGAGGCAGTGCATGCATGGCGCAATGCACTGACGGGGGCCCCCCTGAACCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGCACAGGTGGTGGCCATCGCCAGCAATATTGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGCGCCAGGCCCATGGCCTGACCCCGGAGCAGGTCGTGGCCATCGCCAGCAATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGCACAGGTGGTGGCCATCGCCAGCAATATTGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTCGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGACCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGACCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATGGCGGCGGCAGGCCGGCGCTGGAGAGCATTGTTGCCCAGTTATCTCGCCCTGATCCGGCGTTGGCCGCGTTAACCAACGACCACCTCGTCGCCGGATCCCGCGGCCGCGCCCCCCCGACCGATGTCAGCCTGGGGGACGAGCTCCACTTAGACGGCGAGGACGTGGCGATGGCGCATGCCGACGCGCTAGACGATTTCGATCTGGACATGTTGGGGGACGGGGATTCCCCGGGTCCGGGATTTACCCCCCACGACTCCGCCCCCTACGGCGCTCTGGATATGGCCGACTTCGAGTTTGAGCAGATGTTTACCGATGCCCTTGGAATTGACGAGTACGGTGGCGGCCGCGACTACAAGGACGACGATGACAAG >TALE13 Δ1-13 VP16(SEQ ID NO: 316)MVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQLFRRVGVTELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFADSLERDLDAPSPTHEGDQRRASSRKRSRSDRAVTGPSAQQSFEVRAPEQRDALHLPLSWRVKRPRTSIGGGLPDPGTPTAADLAASSTVMREQDEDPFAGAADDFPAFNEEELAWLMELLPQDRGRAPPTDVSLGDELHLDGEDVAMAHADALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGGGRDYKDDDDK >TALE13 Δ1-13 VP16 (SEQ ID NO: 317)ATGGTGGATCTACGCACGCTCGGCTACAGCCAGCAGCAACAGGAGAAGATCAAACCGAAGGTTCGTTCGACAGTGGCGCAGCACCACGAGGCACTGGTCGGCCATGGGTTTACACACGCGCACATCGTTGCGCTCAGCCAACACCCGGCAGCGTTAGGGACCGTCGCTGTCAAGTATCAGGACATGATCGCAGCGTTGCCAGAGGCGACACACGAAGCGATCGTTGGCGTCGGCAAACAGTGGTCCGGCGCACGCGCCCTGGAGGCCTTGCTCACGGTGGCGGGAGAGTTGAGAGGTCCACCGTTACAGTTGGACACAGGCCAACTTCTCAAGATTGCAAAACGTGGCGGCGTGACCGCAGTGGAGGCAGTGCATGCATGGCGCAATGCACTGACGGGGGCCCCCCTGAACGCGTTAACCAACGACCACCTCGTCGCCTTGGCCTGCCTCGGCGGACGTCCTGCGCTGGATGCAGTGAAAAAGGGATTGCCGCACGCGCCGGCCTTGATCAAAAGAACCAATCGCCGTATTCCCGAACGCACATCCCATCGCGTTGCCGACCACGCGCAAGTGGTTCGCGTGCTGGGTTTTTTCCAGTGCCACTCCCACCCAGCGCAAGCATTTGATGACGCCATGACGCAGTTCGGGATGAGCAGGCACGGGTTGTTACAGCTCTTTCGCAGAGTGGGCGTCACCGAACTCGAAGCCCGCAGTGGAACGCTCCCCCCAGCCTCGCAGCGTTGGGACCGTATCCTCCAGGCATCAGGGATGAAAAGGGCCAAACCGTCCCCTACTTCAACTCAAACGCCGGACCAGGCGTCTTTGCATGCATTCGCCGATTCGCTGGAGCGTGACCTTGATGCGCCCAGCCCAACGCACGAGGGAGATCAGAGGCGGGCAAGCAGCCGTAAACGGTCCCGATCGGATCGTGCTGTCACCGGTCCCTCCGCACAGCAATCGTTCGAGGTGCGCGCTCCCGAACAGCGCGATGCGCTGCATTTGCCCCTCAGTTGGAGGGTAAAACGCCCGCGTACCAGTATCGGGGGCGGCCTCCCGGATCCTGGTACGCCCACGGCTGCCGACCTGGCAGCGTCCAGCACCGTGATGCGGGAACAAGATGAGGACCCCTTCGCAGGGGCAGCGGATGATTTCCCGGCATTCAACGAAGAGGAGCTCGCATGGTTGATGGAGCTATTGCCTCAGGACCGCGGCCGCGCCCCCCCGACCGATGTCAGCCTGGGGGACGAGCTCCACTTAGACGGCGAGGACGTGGCGATGGCGCATGCCGACGCGCTAGACGATTTCGATCTGGACATGTTGGGGGACGGGGATTCCCCGGGTCCGGGATTTACCCCCCACGACTCCGCCCCCTACGGCGCTCTGGATATGGCCGACTTCGAGTTTGAGCAGATGTTTACCGATGCCCTTGGAATTGACGAGTACGGTGGCGGCCGCGACTACAAGGACGACGATGACAAG

4. Miscellaneous DNA Sequences

Donor Used for the Experiment Described in FIG. 37 (SEQ ID NO:318)

AGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCAGAATTAACCCTCACTAAAGGGACTAGTCCTGCAGGTTTAAACGAATTCGCCCTTGATACTTATTAACCATACCTTGGAGGGGAAATCACACATGAAAAGTGTCATTTCTTTACTAATCATATTCATGTCTTTTCTCCCCATAGCAAGACAAAGACCTGTTTTAAACACATTTACAACCTATATGTTGCCTTGTACTAGGTAAAAAGTTGTACATTTCTGAAATAATTTTGGTATTTCTGTTCAGATCACTAAACTCAAGAATCAGCAATTCTCTGAGGCTTTCTTTTAAATATACATAAGGAACTTTCGGAGTGAAGGGAGAGTTTGTCAATAACTTGATGCATGTGAAGGGGAGATAAAAAGGTTGCTATTTTTCATCAACATATTTTGATTTGGCTTTCTATAATTGATGGGCTTAAAAGATCTAATCTACTTTAAACAGATGCCAAATAAATGGATGAATCTTAGACCCTCTATAACAGTAACTTCCTTTTAAAAAAGACCTCTCCCACCCCACCCCCAGCCCAGGCTGTGTATGAAAACTAAGCCATGTGCACAACTCTGACTGGGTCACCAGCCCACTTGAGTCCGTGTCACAAGCCCACAGATATTTCCTGCTCCCCAGTGGATCGGGTGTAAACTGAGCTTGCTCGCTCGGGAGCCTCTTGCTGGAAAATAGAACAGCATTTGCAGAAGCGTTTGGCAATGTGCTTTTGGAAGAAGACTAAGAGGTAGTTTCTGAACTTCTCCCCGACAAAGGCATAGATGATGGGGTTGATGCAGCAGTGCGTCATCCCAAGAGTCTCTGTCACCTGCATAGCTTGGTCCAACCTGTTAGAGCTACTGCAATTATTCAGGCCAAAGAATTCCTGGAAGGTGTTCAGGAGAAGGACAATGTTGTAGGGAGCCCAGAAGAGAAAATAAACAATCATGATGGTGAAGATAAGCCTCACAGCCCTGTGCCTCTTCTTCTCATTTCGACACCGAAGCAGAGTTTTTAGGATTCCCGAGTAGCAGATGACCATGACAAGCAGCGGCAGGACCAGCCCCAAGATGACTATCTTTAATGTCTGGAAATTCTTCCAGAATTGATACTGACTGTATGGAAAATGAGAGCTGCAGGTGTAATGAAGACCTTCTTTTTGAGATCTGGTAAAGATGATTCCTGGGAGAGACGCAAACACAGCCACCACCCAAGTGATCACACTTGTCACCACCCCAAAGGTGACCGTCCTGGCTTTTAAAGCAAACACAGCATGGACGACAGCCAGGTACCTATCGATTGTCAGGAGGATGATGAAGAAGATTCCAGAGAAGAAGCCTATAAAATAGAGCCCTGTCAAGAGTTGACACATTGTATTTCCAAAGTCCCACTGGGCGGCAGCATAGTGAGCCCAGAAGGGGACAGTAAGAAGGAAAAACAGGTCAGAGATGGCCAGGTTGAGCAGGTAGATGTCAGTCATGCTCTTCAGCCTTTTGCAGTTTTCTAGACGAGGCATCCAGTCCAGACGCCATCAGGGCATACTCACTGATCTAGATGAGGATGACCAGCATGTTGCCCACAAAACCAAAGATGAACACCAGTGAGTAGAGCGGAGGCAGGAGGCGGGCTGCGATTTGCTTCACATTGATTTTTTGGCAGGGCTCCGATGTATAATAATTGATGTCATAGATTGGACTTGACACTTGATAATCCATCTTGTTCCACCCTGTGCATAAATAAAAAGTGATCTTTTATAAAGTCCTAGAATGTATTTAGTTGCCCTCCATGAATGCAAACTGTTTTATACATCAATAGGTTTTTAATTGCCTACATAGATGTCTACATTGAATTAACTCTCTTTTTGGCCAAGCAATGAAGTTTTGTAGTGAAGGGAAGGTTTGCTGCTAGCTTCCCTGTCCACTAGATGGAGAGCTTGGCTCTGTTGGGGGAATTCATGAAAGCACCATCTCACCAAATAAAATCTTGTGCTCTATAGCACCATGGAGTGAATGAAGCTTTGACAACAATTAAGGGCGAATTCGCGGCCGCTAAATTCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTATACGTACGGCAGTTTAAGGTTTACACCTATAAAAGAGAGAGCCGTTATCGTCTGTTTGTGGATGTACAGAGTGATATTATTGACACGCCGGGGCGACGGATGGTGATCCCCCTGGCCAGTGCACGTCTGCTGTCAGATAAAGTCTCCCGTGAACTTTACCCGGTGGTGCATATCGGGGATGAAAGCTGGCGCATGATGACCACCGATATGGCCAGTGTGCCGGTCTCCGTTATCGGGGAAGAAGTGGCTGATCTCAGCCACCGCGAAAATGACATCAAAAACGCCATTAACCTGATGTTCTGGGGAATATAAATGTCAGGCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTCACGTAGAAAGCCAGTCCGCAGAAACGGTGCTGACCCCGGATGAATGTCAGCTACTGGGCTATCTGGACAAGGGAAAACGCAAGCGCAAAGAGAAAGCAGGTAGCTTGCAGTGGGCTTACATGGCGATAGCTAGACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAATTGCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGGATGGCTTTCTTGCCGCCAAGGATCTGATGGCGCAGGGGATCAAGCTCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGGCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGAGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAATTATTAACGCTTACAATTTCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAG

TALE13 Reporter Construct (TALE13 BINDING SITES AND SV40 PROMOTERUNDERLINED) (SEQ ID NO:319):

GGTACCGAGCTCTTACGCGTGCTAGTATAAATACCTTCTGCCTTACTAGTATAAATACCTTCTGCCTTGCTAGCTCGAGATCTGCGATCTGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATCGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTGGCATTCCGGTACTGTTGGTAAAGCCACCATGGAAGACGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCGCTGGAAGATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCTGGTTCCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGGACATCACTTACGCTGAGTACTTCGAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTGAATACAAATCACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGCCGGTGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTTATAATGAACGTGAATTGCTCAACAGTATGGGCATTTCGCAGCCTACCGTGGTGTTCGTTTCCAAAAAGGGGTTGCAAAAAATTTTGAACGTGCAAAAAAAGCTCCCAATCATCCAAAAAATTATTATCATGGATTCTAAAACGGATTACCAGGGATTTCAGTCGATGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGAATACGATTTTGTGCCAGAGTCCTTCGATAGGGACAAGACAATTGCACTGATCATGAACTCCTCTGGATCTACTGGTCTGCCTAAAGGTGTCGCTCTGCCTCATAGAACTGCCTGCGTGAGATTCTCGCATGCCAGAGATCCTATTTTTGGCAATCAAATCATTCCGGATACTGCGATTTTAAGTGTTGTTCCATTCCATCACGGTTTTGGAATGTTTACTACACTCGGATATTTGATATGTGGATTTCGAGTCGTCTTAATGTATAGATTTGAAGAAGAGCTGTTTCTGAGGAGCCTTCAGGATTACAAGATTCAAAGTGCGCTGCTGGTGCCAACCCTATTCTCCTTCTTCGCCAAAAGCACTCTGATTGACAAATACGATTTATCTAATTTACACGAAATTGCTTCTGGTGGCGCTCCCCTCTCTAAGGAAGTCGGGGAAGCGGTTGCCAAGAGGTTCCATCTGCCAGGTATCAGGCAAGGATATGGGCTCACTGAGACTACATCAGCTATTCTGATTACACCCGAGGGGGATGATAAACCGGGCGCGGTCGGTAAAGTTGTTCCATTTTTTGAAGCGAAGGTTGTGGATCTGGATACCGGGAAAACGCTGGGCGTTAATCAAAGAGGCGAACTGTGTGTGAGAGGTCCTATGATTATGTCCGGTTATGTAAACAATCCGGAAGCGACCAACGCCTTGATTGACAAGGATGGATGGCTACATTCTGGAGACATAGCTTACTGGGACGAAGACGAACACTTCTTCATCGTTGACCGCCTGAAGTCTCTGATTAAGTACAAAGGCTATCAGGTGGCTCCCGCTGAATTGGAATCCATCTTGCTCCAACACCCCAACATCTTCGACGCAGGTGTCGCAGGTCTTCCCGACGATGACGCCGGTGAACTTCCCGCCGCCGTTGTTGTTTTGGAGCACGGAAAGACGATGACGGAAAAAGAGATCGTGGATTACGTCGCCAGTCAAGTAACAACCGCGAAAAAGTTGCGCGGAGGAGTTGTGTTTGTGGACGAAGTACCGAAAGGTCTTACCGGAAAACTCGACGCAAGAAAAATCAGAGAGATCCTCATAAAGGCCAAGAAGGGCGGAAAGATCGCCGTGTAATTCTAGAGTCGGGGCGGCCGGCCGCTTCGAGCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTAAAATCGATAAGGATCCGTCGACCGATGCCCTTGAGAGCCTTCAACCCAGTCAGCTCCTTCCGGTGGGCGCGGGGCATGACTATCGTCGCCGCACTTATGACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCCCAAGCTACCATGATAAGTAAGTAATATTAAGGTACGGGAGGTACTTGGAGCGGCCGCAATAAAATATCTTTATTTTCATTACATCTGTGTGTTGGTTTTTTGTGTGAATCGATAGTACTAACATACGCTCTCCATCAAAACAAAACGAAACAAAACAAACTAGCAAAATAGGCTGTCCCCAGTGCAAGTGCAGGTGCCAGAACATTTCTCTATCGATA

DNA Sequence of TALE13 (SEQ ID NO:320):

GTGGATCTACGCACGCTCGGCTACAGCCAGCAGCAACAGGAGAAGATCAAACCGAAGGTTCGTTCGACAGTGGCGCAGCACCACGAGGCACTGGTCGGCCATGGGTTTACACACGCGCACATCGTTGCGCTCAGCCAACACCCGGCAGCGTTAGGGACCGTCGCTGTCAAGTATCAGGACATGATCGCAGCGTTGCCAGAGGCGACACACGAAGCGATCGTTGGCGTCGGCAAACAGTGGTCCGGCGCACGCGCCCTGGAGGCCTTGCTCACGGTGGCGGGAGAGTTGAGAGGTCCACCGTTACAGTTGGACACAGGCCAACTTCTCAAGATTGCAAAACGTGGCGGCGTGACCGCAGTGGAGGCAGTGCATGCATGGCGCAATGCACTGACGGGTGCCCCCCTGAACCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGCACAGGTGGTGGCCATCGCCAGCAATATTGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGCGCCAGGCCCATGGCCTGACCCCGGAGCAGGTCGTGGCCATCGCCAGCAATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGCACAGGTGGTGGCCATCGCCAGCAATATTGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTCGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGACCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGACCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTGCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCATGGCCTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATGGCGGCGGCAGGCCGGCGCTGGAGAGCATTGTTGCCCAGTTATCTCGCCCTGATCCGGCGTTGGCCGCGTTGACCAACGACCACCTCGTCGCCTTGGCCTGCCTCGGCGGACGTCCTGCGCTGGATGCAGTGAAAAAGGGATTGCCGCACGCGCCGGCCTTGATCAAAAGAACCAATCGCCGTATTCCCGAACGCACATCCCATCGCGTTGCCGACCACGCGCAAGTGGTTCGCGTGCTGGGTTTTTTCCAGTGCCACTCCCACCCAGCGCAAGCATTTGATGACGCCATGACGCAGTTCGGGATGAGCAGGCACGGGTTGTTACAGCTCTTTCGCAGAGTGGGCGTCACCGAACTCGAAGCCCGCAGTGGAACGCTCCCCCCAGCCTCGCAGCGTTGGGACCGTATCCTCCAGGCATCAGGGATGAAAAGGGCCAAACCGTCCCCTACTTCAACTCAAACGCCGGACCAGGCGTCTTTGCATGCATTCGCCGATTCGCTGGAGCGTGACCTTGATGCGCCCAGCCCAACGCACGAGGGAGATCAGAGGCGGGCAAGCAGCCGTAAACGGTCCCGATCGGATCGTGCTGTCACCGGTCCCTCCGCACAGCAATCGTTCGAGGTGCGCGCTCCCGAACAGCGCGATGCGCTGCATTTGCCCCTCAGTTGGAGGGTAAAACGCCCGCGTACCAGTATCGGGGGCGGCCTCCCGGATCCTGGTACGCCCACGGCTGCCGACCTGGCAGCGTCCAGCACCGTGATGCGGGAACAAGATGAGGACCCCTTCGCAGGGGCAGCGGATGATTTCCCGGCATTCAACGAAGAGGAGCTCGCATGGTTGATGGAGCTATTGCCTCAG

Protein and Gene Sequences of TALEs VEGF-1 and CCR5-1

>VEGF-1 (SEQ ID NO: 321)VDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMS >VEGF-1 (SEQ ID NO: 322)GTGGATCTACGCACGCTCGGCTACAGCCAGCAGCAACAGGAGAAGATCAAACCGAAGGTTCGTTCGACAGTGGCGCAGCACCACGAGGCACTGGTCGGCCATGGGTTTACACACGCGCACATCGTTGCGCTCAGCCAACACCCGGCAGCGTTAGGGACCGTCGCTGTCAAGTATCAGGACATGATCGCAGCGTTGCCAGAGGCGACACACGAAGCGATCGTTGGCGTCGGCAAACAGTGGTCCGGCGCACGCGCCCTGGAGGCCTTGCTCACGGTGGCGGGAGAGTTGAGAGGTCCACCGTTACAGTTGGACACAGGCCAACTTCTCAAGATTGCAAAACGTGGCGGCGTGACCGCAGTGGAGGCAGTGCATGCATGGCGCAATGCACTGACGGGGGCCCCCCTGAACCTGACGCCTCAACAGGTCGTCGCGATAGCGTCTAATAATGGAGGAAAGCAAGCTCTGGAAACCGTCCAGCGACTCCTTCCGGTTCTGTGCCAGGCTCATGGTCTGACTCCGCAGCAAGTCGTTGCTATAGCGTCCAACATCGGAGGCAAACAGGCCCTGGAGACCGTGCAGCGGTTGTTGCCTGTGCTTTGCCAAGCCCACGGGCTTACGCCTGAGCAAGTGGTGGCGATTGCCAGTAACAACGGCGGCAAACAAGCCCTTGAGACTGTGCAGAGGCTCTTGCCGGTACTCTGCCAAGCACACGGCTTGACCCCCGAGCAGGTTGTAGCCATAGCTAGTCACGACGGGGGTAAGCAAGCGTTGGAAACGGTGCAAGCACTTCTCCCCGTTCTCTGTCAAGCGCATGGACTTACCCCGGAACAGGTGGTCGCCATTGCAAGCCATGATGGGGGTAAGCAAGCGTTGGAAACGGTGCAAGCACTTCTCCCCGTTCTCTGTCAAGCGCATGGACTTACCCCGGAACAGGTGGTCGCCATTGCAAGCCATGATGGAGGAAAGCAGGCGCTCGAAACAGTCCAGGCACTTTTGCCCGTACTTTGTCAAGCTCACGGTCTCACCCCGGAACAGGTGGTAGCCATTGCATCTAACGGAGGGGGCAAACAAGCCTTGGAGACAGTGCAAAGGCTCCTGCCAGTGCTCTGCCAGGCTCATGGTTTGACACCCGAACAGGTAGTTGCAATAGCGAGTCATGATGGCGGAAAGCAAGCTCTTGAAACTGTGCAGCGGCTGTTGCCTGTACTGTGTCAAGCCCACGGGCTGACACCGGAACAAGTTGTAGCGATCGCTAGCCACGATGGCGGGAAACAAGCTCTGGAAACGGTACAGAGACTCCTCCCAGTGCTTTGTCAGGCACACGGCCTCACGCCAGAGCAGGTTGTCGCCATCGCGTCACATGATGGGGGCAAACAAGCCTTGGAGACAGTGCAAAGGCTCCTGCCAGTGCTCTGCCAGGCTCATGGTTTGACACCCGAACAGGTAGTTGCAATAGCGAGTCATGATGGCGGAAAGCAAGCTCTTGAAACTGTGCAGCGGCTGTTGCCTGTACTGTGTCAAGCCCACGGGCTGACACCGGAACAAGTTGTAGCGATCGCTAGCCACGATGGCGGGAAACAAGCTCTGGAAACGGTACAGAGACTCCTCCCAGTGCTTTGTCAGGCACACGGCCTCACGCCAGAGCAGGTTGTCGCCATCGCGTCAAACGGTGGAGGGAAACAAGCGCTCGAAACCGTGCAAAGGTTGCTCCCCGTTCTCTGTCAGGCGCACGGTCTTACGCCACAACAGGTGGTGGCGATTGCATCTAATGGAGGCGGACGCCCTGCCTTGGAGAGCATTGTGGCCCAGCTGTCCAGGCCGGACCCTGCCCTGGCCGCGTTAACCAACGACCACCTCGTCGCCTTGGCCTGCCTCGGCGGACGTCCTGCGCTGGATGCAGTGAAAAAGGGATTGCCGCACGCGCCGGCCTTGATCAAAAGAACCAATCGCCGTATTCCCGAACGCACATCCCATCGCGTTGCCGACCACGCGCAAGTGGTTCGCGTGCTGGGTTTTTTCCAGTGCCACTCCCACCCAGCGCAAGCATTTGATGACGCCATGACGCAGTTCGGGATGAGC >CCR5-1 (SEQ ID NO: 323)VDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNKGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMS >CCR5-1(SEQ ID NO: 324)GTGGATCTACGCACGCTCGGCTACAGCCAGCAGCAACAGGAGAAGATCAAACCGAAGGTTCGTTCGACAGTGGCGCAGCACCACGAGGCACTGGTCGGCCATGGGTTTACACACGCGCACATCGTTGCGCTCAGCCAACACCCGGCAGCGTTAGGGACCGTCGCTGTCAAGTATCAGGACATGATCGCAGCGTTGCCAGAGGCGACACACGAAGCGATCGTTGGCGTCGGCAAACAGTGGTCCGGCGCACGCGCCCTGGAGGCCTTGCTCACGGTGGCGGGAGAGTTGAGAGGTCCACCGTTACAGTTGGACACAGGCCAACTTCTCAAGATTGCAAAACGTGGCGGCGTGACCGCAGTGGAGGCAGTGCATGCATGGCGCAATGCACTGACGGGGGCCCCCCTGAACCTTACACCCGAGCAAGTAGTGGCTATTGCGAGTAATAAAGGGGGTAAGCAAGCGTTGGAAACGGTGCAAGCACTTCTCCCCGTTCTCTGTCAAGCGCATGGACTTACCCCGGAACAGGTGGTCGCCATTGCAAGCCATGATGGAGGAAAGCAGGCGCTCGAAACAGTCCAGGCACTTTTGCCCGTACTTTGTCAAGCTCACGGTCTCACCCCGGAACAGGTGGTAGCCATTGCATCTAACGGAGGGGGCAAACAAGCCTTGGAGACAGTGCAAAGGCTCCTGCCAGTGCTCTGCCAGGCTCATGGTTTGACACCCGAACAGGTAGTTGCAATAGCGAGTCATGATGGCGGAAAGCAAGCTCTTGAAACTGTGCAGCGGCTGTTGCCTGTACTGTGTCAAGCCCACGGGCTGACACCGGAACAAGTTGTAGCGATCGCTAGCAACGGCGGAGGTAAGCAAGCATTGGAAACGGTTCAGGCCCTGTTGCCTGTACTTTGCCAGGCGCACGGTCTGACACCTGAGCAGGTTGTCGCCATCGCTAGCAACGGAGGTGGGAAACAGGCACTTGAAACTGTGCAGAGGCTTCTGCCGGTGCTGTGCCAAGCGCATGGCCTTACACCCGAGCAAGTAGTGGCTATTGCGAGTCATGATGGAGGCAAGCAAGCGCTGGAGACTGTCCAACGACTTCTTCCGGTCTTGTGTCAGGCACATGGATTGACCCCTCAACAAGTCGTGGCGATAGCTAGCAACATCGGAGGCAAACAGGCCCTGGAGACCGTGCAGCGGTTGTTGCCTGTGCTTTGCCAAGCCCACGGGCTTACGCCTGAGCAAGTGGTGGCGATTGCCAGTAACAACGGGGGCAAACAAGCCTTGGAGACAGTGCAAAGGCTCCTGCCAGTGCTCTGCCAGGCTCATGGTTTGACACCCGAACAGGTAGTTGCAATAGCGAGTCATGATGGCGGAAAGCAAGCTCTTGAAACTGTGCAGCGGCTGTTGCCTGTACTGTGTCAAGCCCACGGGCTGACACCGGAACAAGTTGTAGCGATCGCTAGCCACGATGGCGGGAAACAAGCTCTGGAAACGGTACAGAGACTCCTCCCAGTGCTTTGTCAGGCACACGGCCTCACGCCAGAGCAGGTTGTCGCCATCGCGTCAAACGGTGGAGGGAAACAAGCGCTCGAAACCGTGCAAAGGTTGCTCCCCGTTCTCTGTCAGGCGCACGGTCTTACGCCACAACAGGTGGTGGCGATTGCATCTAATGGAGGCGGACGCCCTGCCTTGGAGAGCATTGTGGCCCAGCTGTCCAGGCCGGACCCTGCCCTGGCCGCGTTAACCAACGACCACCTCGTCGCCTTGGCCTGCCTCGGCGGACGTCCTGCGCTGGATGCAGTGAAAAAGGGATTGCCGCACGCGCCGGCCTTGATCAAAAGAACCAATCGCCGTATTCCCGAACGCACATCCCATCGCGTTGCCGACCACGCGCAAGTGGTTCGCGTGCTGGGTTTTTTCCAGTGCCACTCCCACCCAGCGCAAGCATTTGATGACGCCATGACGCAGTTCGGGATGAGC

Gene Sequences of AAVS1-Specific TALENs

101077 ORF (TALE region underlined) (SEQ ID NO: 325):MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS101079 ORF (TALE region underlined) (SEQ ID NO: 326):MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRSSequence of ben-1 Specific TALENs ORFS:

101318 (SEQ ID NO: 327)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS 101321(SEQ ID NO: 328)MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS pZMt-101380 (SEQ ID NO: 444)ctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatacgcgtaccgctagccaggaagagtttgtagaaacgcaaaaaggccatccgtcaggatggccttctgcttagtttgatgcctggcagtttatggcgggcgtcctgcccgccaccctccgggccgttgcttcacaacgttcaaatccgctcccggcggatttgtcctactcaggagagcgttcaccgacaaacaacagataaaacgaaaggcccagtcttccgactgagcctttcgttttatttgatgcctggcagttccctactctcgcgttaacgctagcatggatgttttcccagtcacgacgttgtaaaacgacggccagtcttaagctcgggccccaaataatgatttattttgactgatagtgacctgttcgttgcaacaaattgatgagcaatgcttttttataatgccaactttgtacaaaaaagcaggctccgaattcgcccttttaattaatgcagtgcagcgtgacccggtcgtgcccctctctagagataatgagcattgcatgtctaagttataaaaaattaccacatattttttttgtcacacttgtttgaagtgcagtttatctatctttatacatatatttaaactttactctacgaataatataatctatagtactacaataatatcagtgtttagagaatcatataaatgaacagttagacatggtctaaaggacaattgagtattttgacaacaggactctacagttttatctttttagtgtgcatgtgttctcctttttttttgcaaatagcttcacctatataatacttcatccattttattagtacatccatttagggtttagggttaatggtttttatagactaatttttttagtacatctattttattctattttagcctctaaattaagaaaactaaaactctattttagtttttttatttaataatttagatataaaatagaataaaataaagtgactaaaaattaaacaaataccctttaagaaattaaaaaaactaaggaaacatttttcttgtttcgagtagataatgccagcctgttaaacgccgtcgacgagtctaacggacaccaaccagcgaaccagcagcgtcgcgtcgggccaagcgaagcagacggcacggcatctctgtcgctgcctctggacccctctcgagagttccgctccaccgttggacttgctccgctgtcggcatccagaaattgcgtggcggagcggcagacgtgagccggcacggcaggcggcctcctcctcctctcacggcaccggcagctacgggggattcctttcccaccgctccttcgctttcccttcctcgcccgccgtaataaatagacaccccctccacaccctctttccccaacctcgtgttgttcggagcgcacacacacacaaccagatctcccccaaatccacccgtcggcacctccgcttcaaggtacgccgctcgtcctccccccccccccctctctaccttctctagatcggcgttccggtccatggttagggcccggtagttctacttctgttcatgtttgtgttagatccgtgtttgtgttagatccgtgctgctagcgttcgtacacggatgcgacctgtacgtcagacacgttctgattgctaacttgccagtgtttctctttggggaatcctgggatggctctagccgttccgcagacgggatcgatttcatgattttttttgtttcgttgcatagggtttggtttgcccttttcctttatttcaatatatgccgtgcacttgtttgtcgggtcatcttttcatgcttttttttgtcttggttgtgatgatgtggtctggttgggcggtcgttctagatcggagtagaattctgtttcaaactacctggtggatttattaattttggatctgtatgtgtgtgccatacatattcatagttacgaattgaagatgatggatggaaatatcgatctaggataggtatacatgttgatgcgggttttactgatgcatatacagagatgctttttgttcgcttggttgtgatgatgtggtgtggttgggcggtcgttcattcgttctagatcggagtagaatactgtttcaaactacctggtgtatttattaattttggaactgtatgtgtgtgtcatacatcttcatagttacgagtttaagatggatggaaatatcgatctaggataggtatacatgttgatgtgggttttactgatgcatatacatgatggcatatgcagcatctattcatatgctctaaccttgagtacctatctattataataaacaagtatgttttataattattttgatcttgatatacttggatgatggcatatgcagcagctatatgtggatttttttagccctgccttcatacgctatttatttgcttggtactgtttcttttgtcgatgctcaccctgttgtttggtgttacttctgcaggactagtccagtgtggtggaattcgccatggactacaaagaccatgacggtgattataaagatcatgacatcgattacaaggatgacgatgacaagatggcccccaagaagaagaggaaggtgggcattcacggggtacctatggtggacttgaggacactcggttattcgcaacagcaacaggagaaaatcaagcctaaggtcaggagcaccgtcgcgcaacaccacgaggcgcttgtggggcatggcttcactcatgcgcatattgtcgcgctttcacagcaccctgcggcgcttgggacggtggctgtcaaataccaagatatgattgcggccctgcccgaagccacgcacgaggcaattgtaggggtcggtaaacagtggtcgggagcgcgagcacttgaggcgctgctgactgtggcgggtgagcttagggggcctccgctccagctcgacaccgggcagctgctgaagatcgcgaagagagggggagtaacagcggtagaggcagtgcacgcctggcgcaatgcgctcaccggggcccccttgaacctgaccccagaccaggtagtcgcaatcgcgtcgcatgacgggggaaagcaagccctggaaaccgtgcaaaggttgttgccggtcctttgtcaagaccacggccttacaccggagcaagtcgtggccattgcatcacatgacggtggcaaacaggctcttgagacggttcagagacttctcccagttctctgtcaagcccacgggctgactcccgatcaagttgtagcgattgcgagcaatgggggagggaaacaagcattggagactgtccaacggctccttcccgtgttgtgtcaagcccacggtttgacgcctgcacaagtggtcgccatcgcctccaatattggcggtaagcaggcgctggaaacagtacagcgcctgctgcctgtactgtgccaggatcatggactcaccccagaccaggtagtcgcaatcgcgtcgcatgacgggggaaagcaagccctggaaaccgtgcaaaggttgttgccggtcctttgtcaagaccacggccttacaccggatcaagtcgtggccattgcaaataataacggtggcaaacaggctcttgagacggttcagagacttctcccagttctctgtcaagcccacgggctgactcccgatcaagttgtagcgattgcgagcaacatcggagggaaacaagcattggagactgtccaacggctccttcccgtgttgtgtcaagcccacggtttgacgcctgcacaagtggtcgccatcgcctcccacgacggcggtaagcaggcgctggaaacagtacagcgcctgctgcctgtactgtgccaggatcatgggctgaccccagaccaggtagtcgcaatcgccaacaataacgggggaaagcaagccctggaaaccgtgcaaaggttgttgccggtcctttgtcaagaccacggccttacaccggagcaagtcgtggccattgcatcaaatatcggtggcaaacaggctcttgagacggttcagagacttctcccagttctctgtcaagcccacgggctgactcccgatcaagttgtagcgattgcgaataacaatggagggaaacaagcattggagactgtccaacggctccttcccgtgttgtgtcaagcccacggtttgacgcctgcacaagtggtcgccatcgccaacaacaacggcggtaagcaggcgctggaaacagtacagcgcctgctgcctgtactgtgccaggatcatggtttgaccccagaccaggtagtcgcaatcgcgtcgaacattgggggaaagcaagccctggaaaccgtgcaaaggttgttgccggtcctttgtcaagaccacggccttacaccggatcaagtcgtggccattgcaaataataacggtggcaaacaggctcttgagacggttcagagacttctcccagttctctgtcaagcccacgggctgactcccgatcaagttgtagcgattgcgaataacaatggagggaaacaagcattggagactgtccaacggctccttcccgtgttgtgtcaagcccacggtttgacgcctgcacaagtggtcgccatcgcctccaatattggcggtaagcaggcgctggaaacagtacagcgcctgctgcctgtactgtgccaggatcatggcctgacacccgaacaggtggtcgccattgctagcaacgggggaggacggccagccttggagtccatcgtagcccaattgtccaggcccgatcccgcgttggctgcgttaacgaatgaccatctggtggcgttggcatgtcttggtggacgacccgcgctcgatgcagtcaaaaagggtctgcctcatgctcccgcattgatcaaaagaaccaaccggcggattcccgagagaacttcccatcgagtcgcgggatcccagctggttaaatcagaactcgaagaaaaaaagagcgagctgcggcataaactcaaatatgtccctcatgagtacatagaactgattgaaatcgcccgcaattccacccaggatcggattcttgaaatgaaagtgatggaattttttatgaaagtttacggctatcgcgggaagcaccttggggggtcgcggaagccggacggtgctatttacactgtcggttccccgatcgattatggcgtaattgttgacacgaaagcatattcgggtgggtataatcttcctattggtcaggctgatgagatgcagcggtacgttgaagagaatcagacgcggaacaagcatattaacccaaatgagtggtggaaggtgtatccatcatcggtcaccgaatttaagttcttgtttgtgtcgggccactttaaggggaactacaaggcccaacttaccaggttgaatcacataaccaactgtaacggagctgttctgtcagtagaagagctgttgataggcggggaaatgattaaagcaggtacattaacgttggaggaagtacgccgcaagtttaataacggcgagattaactttagatctgagacctgataaacaaacacacggtctcctcgagctcgcagatcgttcaacatctggcaataaagtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggtttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatccgataagcttaagggcgaattcgacccagctttcttgtacaaagttggcattataaaaaataattgctcatcaatttgttgcaacgaacaggtcactatcagtcaaaataaaatcattatttgccatccagctgatatcccctatagtgagtcgtattacatggtcatagctgtttcctggcagctctggcccgtgtctcaaaatctctgatgttacattgcacaagataaaaatatatcatcatgcctcctctagaccagccaggacagaaatgcctcgacttcgctgctgcccaaggttgccgggtgacgcacaccgtggaaacggatgaaggcacgaacccagtggacataagcctgttcggttcgtaagctgtaatgcaagtagcgtatgcgctcacgcaactggtccagaaccttgaccgaacgcagcggtggtaacggcgcagtggcggttttcatggcttgttatgactgtttttttggggtacagtctatgcctcgggcatccaagcagcaagcgcgttacgccgtgggtcgatgtttgatgttatggagcagcaacgatgttacgcagcagggcagtcgccctaaaacaaagttaaacatcatgagggaagcggtgatcgccgaagtatcgactcaactatcagaggtagttggcgtcatcgagcgccatctcgaaccgacgttgctggccgtacatttgtacggctccgcagtggatggcggcctgaagccacacagtgatattgatttgctggttacggtgaccgtaaggcttgatgaaacaacgcggcgagctttgatcaacgaccttttggaaacttcggcttcccctggagagagcgagattctccgcgctgtagaagtcaccattgttgtgcacgacgacatcattccgtggcgttatccagctaagcgcgaactgcaatttggagaatggcagcgcaatgacattcttgcaggtatcttcgagccagccacgatcgacattgatctggctatcttgctgacaaaagcaagagaacatagcgttgccttggtaggtccagcggcggaggaactctttgatccggttcctgaacaggatctatttgaggcgctaaatgaaaccttaacgctatggaactcgccgcccgactgggctggcgatgagcgaaatgtagtgcttacgttgtcccgcatttggtacagcgcagtaaccggcaaaatcgcgccgaaggatgtcgctgccgactgggcaatggagcgcctgccggcccagtatcagcccgtcatacttgaagctagacaggcttatcttggacaagaagaagatcgcttggcctcgcgcgcagatcagttggaagaatttgtccactacgtgaaaggcgagatcaccaaggtagtcggcaaataaccctcgagccacccatgaccaaaatcccttaacgtgagttacgcgtcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagcattgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgtt

1. An isolated, non-naturally occurring DNA-binding polypeptide comprising: at least one TALE-repeat unit; an N-cap polypeptide; and a C-cap polypeptide, wherein the C-cap polypeptide comprises a fragment of a TALE protein.
 2. The isolated polypeptide of claim 1, wherein at least one TALE-repeat unit comprises an atypical repeat variable di-residue (RVD).
 3. The polypeptide of claim 2, wherein the protein comprises an atypical RVD as shown in Table
 27. 4. The polypeptide of claim 1, wherein the C-cap polypeptide is less than approximately 230 amino acids in length.
 5. The polypeptide of claim 1, wherein the C-cap comprises a TALE repeat domain.
 6. A fusion protein comprising the polypeptide of claim 1 and at least one functional domain.
 7. The fusion protein of claim 6, wherein the functional domain is a transcriptional activator or a transcriptional repressor.
 8. The fusion protein of claim 7, wherein the functional domain comprises a nuclease.
 9. The fusion protein of claim 8, wherein the nuclease comprises at least one cleavage domain or cleavage half-domain from a TypeIIS endonuclease.
 10. A polynucleotide encoding a polypeptide according to claim
 1. 11. A polynucleotide encoding a fusion protein according to claim
 6. 12. A host cell comprising a polypeptide according to claim
 1. 13. A host cell comprising a polynucleotide according to claim
 10. 14. A pharmaceutical composition comprising a polypeptide according to claim
 1. 15. A pharmaceutical composition comprising a polynucleotide according to claim
 10. 16. A method of modulating expression of an endogenous gene in a cell, the method comprising: introducing into the cell a fusion protein according to claim 6, wherein the fusion protein comprises a TALE-repeat domain that binds to a target site in the endogenous gene and further wherein expression of the endogenous gene is modulated.
 17. The method of claim 16, wherein the modulation comprises gene activation.
 18. The method of claim 16, wherein the modulation comprises gene repression or inactivation.
 19. The method of claim 16, wherein the fusion protein comprises a cleavage domain or cleavage half-domain and the endogenous gene is inactivated by cleavage.
 20. The method of claim 19, wherein inactivation occurs via non-homologous end joining (NHEJ).
 21. The method of claim 16, wherein the fusion protein is introduced as a polynucleotide encoding the fusion protein.
 22. A method of modifying a region of interest in the genome of a cell, the method comprising: introducing into the cell at least one fusion protein according to claim 8, wherein the fusion protein comprises a TALE-repeat domain that binds to a target site in the genome of the cell and the fusion protein cleaves the genome in the region of interest.
 23. The method of claim 22, wherein the modifying comprises introducing a deletion in the region of interest.
 24. The method of claim 18, wherein the modifying comprises introducing an exogenous nucleic acid into the region of interest, the method further comprising introducing the exogenous nucleic acid into the cell, wherein the exogenous nucleic acid is integrated into the region of interest by homologous recombination.
 25. The method of claim 22, wherein the cell is a eukaryotic cell selected from the selected from the group consisting of a plant cell, an animal cell, a fish cell and a yeast cell.
 26. The method of claim 22, wherein the fusion protein is introduced as a polynucleotide encoding the fusion protein. 